Isolated myeloid-like bone marrow cell populations and methods of treatment therewith

ABSTRACT

The present invention provides an isolated myeloid-like bone marrow cell population comprising a majority of cells that are lineage negative, and which express both CD44 antigen and CD11b antigen. These cells have beneficial vasculotrophic and neurotrophic activity when intraocularly administered to the eye of a mammal, particularly a mammal suffering from an ocular degenerative disease. The myeloid-like bone marrow cells are isolated by treating bone marrow cells with an antibody against CD44 (hyaluronic acid receptor), against CD11b, or against both and using flow cytometry to positively select CD44 and/or CD11b expressing cells therefrom. The isolated myeloid-like bone marrow cells of the invention can be transfected with a gene encoding a therapeutically useful protein, for delivering the gene to the retina.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/884,958, filed on Aug. 23, 2007, now U.S. Pat. No. 7,931,891, whichis the National Stage of International Application No.PCT/US2006/006411, filed on Feb. 24, 2006, which claims the benefit ofU.S. Provisional Patent Application No. 60/656,037, filed on Feb. 24,2005, each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

A portion of the work described herein was supported by grant numbersEY11254, EY12598, and EY13916 from the National Institutes of Health.The United States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to isolated, mammalian, bone marrow cells. Moreparticularly the invention is related to isolated bone marrow cellpopulations that have myeloid cell characteristics and are capable ofbeing incorporated into retinal vasculature when intravitreally injectedinto the eye. The invention also relates to methods of treating oculardegenerative diseases by administering isolated bone marrow cells to theeye of a mammal.

BACKGROUND OF THE INVENTION

Age related macular degeneration (ARMD) and diabetic retinopathy (DR)are the leading causes of visual loss in industrialized nations and doso as a result of abnormal retinal neovascularization. Since the retinaconsists of well-defined layers of neuronal, glial, and vascularelements, relatively small disturbances such as those seen in vascularproliferation or edema can lead to significant loss of visual function.Inherited retinal degenerations, such as retinitis pigmentosa (RP), arealso associated with vascular abnormalities, such as arteriolarnarrowing and vascular atrophy. Most inherited human retinaldegenerations specifically affect rod photoreceptors, but there is alsoa concomitant loss of cones, the principal cellular component of themacula, the region of the retina in humans that is responsible forcentral, fine visual acuity. Cone-specific survival factors have beendescribed recently (Mohand-Said et al. 1998, Proc. Natl. Acad. Sci. USA,95: 8357-8362) and may facilitate cone survival in mouse models ofretinal degeneration.

Inherited degenerations of the retina affect as many as 1 in 3500individuals and are characterized by progressive night blindness, visualfield loss, optic nerve atrophy, arteriolar attenuation, alteredvascular permeability and central loss of vision often progressing tocomplete blindness (Heckenlively, J. R., editor, 1988; RetinitisPigmentosa, Philadelphia: JB Lippincott Co.). Molecular genetic analysisof these diseases has identified mutations in over 110 different genesaccounting for only a relatively small percentage of the known affectedindividuals (Humphries et al., 1992, Science 256:804-808; Farrar et al.2002, EMBO J. 21:857-864). Many of these mutations are associated withenzymatic and structural components of the phototransduction machineryincluding rhodopsin, cGMP phosphodiesterase, rds peripherin, and RPE65.Despite these observations, there are still no effective treatments toslow or reverse the progression of these retinal degenerative diseases.Recent advances in gene therapy have led to successful reversal of therds (Ali et al. 2000, Nat. Genet. 25:306-310) and rd (Takahashi et al.1999, J. Virol. 73:7812-7816) phenotypes in mice and the RPE65 phenotypein dogs (Acland et al. 2001, Nat. Genet. 28:92-95) when the wild typetransgene is delivered to photoreceptors or the retinal pigmentedepithelium (RPE) in animals with a specific mutation.

For many years it has been known that a population of stem cells existsin the normal adult circulation and bone marrow. Differentsub-populations of these cells can differentiate along hematopoieticlineage positive (Lin⁺) or lineage negative (Lin⁻) lineages.Furthermore, the lineage negative hematopoietic stem cell (HSC)population has recently been shown to contain endothelial progenitorcells (EPC) capable of forming blood vessels in vitro and in vivo (SeeAsahara et al. 1997, Science 275: 964-7). These cells can participate innormal and pathological postnatal angiogenesis (See Lyden et al. 2001Nat. Med. 7, 1194-201; Kalka et al. 2000, Proc. Natl. Acad. Sci. U.S.A.97:3422-7; and Kocher et al. 2001, Nat. Med. 7: 430-6) as well asdifferentiate into a variety of non-endothelial cell types includinghepatocytes (See Lagasse et al. 2000, Nat. Med. 6:1229-34), microglia(See Priller et al. 2002 Nat. Med. 7:1356-61), cardiomyocytes (See Orlicet al. 2001, Proc. Natl. Acad. Sci. U.S.A. 98: 10344-9) and epithelium(See Lyden et al. 2001, Nat. Med. 7:1194-1201). Although these cellshave been used in several experimental models of angiogenesis, themechanism of EPC targeting to neovasculature is not known, and nostrategy has been identified that will effectively increase the numberof cells that contribute to a particular vasculature.

Hematopoietic stem cells from bone marrow are currently the only type ofstem cell commonly used for therapeutic applications. Bone marrow HSC'shave been used in transplants for over 40 years. Currently, advancedmethods of harvesting purified stem cells are being investigated todevelop therapies for treatment of leukemia, lymphoma, and inheritedblood disorders. Clinical applications of stem cells in humans have beeninvestigated for the treatment of diabetes and advanced kidney cancer inlimited numbers of human patients.

SUMMARY OF THE INVENTION

The present invention provides an isolated myeloid-like bone marrow(MLBM) cell population produced by positively selecting cells thatexpress CD44, CD11b, or both antigens, from bone marrow of a mammal.These cells exhibit beneficial vasculotrophic and neurotrophic activitywhen intraocularly administered to the eye of a mammal, particularly amammal suffering from an ocular degenerative disease. The MLBM cellpopulation of the invention can be isolated by treating bone marrowcells with an antibody against CD44 (hyaluronic acid receptor), anantibody against CD11b, or antibodies against both antigens, andpositively selecting cells that immunoreact with the antibody orantibodies, as the case may be (e.g., using flow cytometry orantibody-coated or bound beads to separate the cells). A majority of thecells of the MLBM cell population of the invention are lineage negativeand express both the CD44 antigen and the CD11b antigen.

The present invention also provides a method of treating vasculotrophicand neurotrophic retinal diseases in a mammal. The method comprisesadministering isolated cells from the MLBM cell population to thediseased eye of a mammal, preferably by intraocular injection.Preferably, the MLBM cell population is autologous to the mammal beingtreated (i.e., the MLBM cell population was isolated from the bonemarrow of the individual mammal to be treated). The present treatmentmethod ameliorates vascular degeneration and degeneration ofphotoreceptor neurons in the retina of a mammal that suffers from anocular disease. The cells are administered in an amount sufficient toretard vascular and neural degeneration in the retina. Beneficially, thecells from the MLBM cell population incorporate into the vasculature ofthe retina and differentiate into endothelial cells, while at the sametime incorporating into the neuronal network and ameliorating thedegeneration of cone cells in the retina. The isolated, mammalian, MLBMcell population, includes cells that selectively target activatedretinal astrocytes when intravitreally injected into the eye, and remainstably incorporated into neovasculature and neuronal network of the eye.Preferably the mammal is a human.

In a preferred embodiment, at least about 75 percent of the cells in theisolated myeloid-like bone marrow cell population express CD44, morepreferably at least about 90 percent.

In one preferred embodiment, cells from the MLBM cell population aretransfected with a therapeutically useful gene. For example, the cellscan be transfected with polynucleotides that operably encode forneurotrophic agents or anti-angiogenic agents that selectively targetneovasculature and inhibit new vessel formation without affectingalready established vessels through a form of cell-based gene therapy.In one embodiment, isolated, MLBM cell population of the inventioninclude a gene encoding an angiogenesis inhibiting peptide. Theangiogenesis inhibiting cells from the MLBM cell population are usefulfor modulating abnormal blood vessel growth in diseases such as ARMD, DRand certain retinal degenerations associated with abnormal vasculature.In another preferred embodiment, the isolated, cells from the MLBM cellpopulation of the present invention are transfected to include a geneencoding a neurotrophic peptide. The neurotrophic transfected MLBM cellsare useful for promoting neuronal rescue in ocular diseases involvingretinal neural degeneration, such as glaucoma, retinitis pigmentosa, andthe like.

A particular advantage of ocular treatments with the isolated MLBM cellpopulation of the present invention is a vasculotrophic and neurotrophicrescue effect observed in eyes intravitreally treated with cells fromthe MLBM cell population. Retinal neurons and photoreceptors,particularly cones, are preserved and some measure of visual functioncan be maintained in eyes treated with cells from the MLBM cellpopulation of the invention.

The present invention also provides a method for isolating amyeloid-like bone marrow cell population from bone marrow by negativecell marker selection. The method comprises contacting a plurality ofbone marrow cells with antibodies specific for Ter119, CD45RB220, andCD3e, removing cells from the plurality of bone marrow cells thatimmunoreact with Ter119, CD45RB220, and CD3e antibodies, and recoveringmyeloid-like bone marrow cells that are deleted in Ter119, CD45RB220,and CD3e-expressing cells. Using this method, a cell population can berecovered in which greater than 90 percent of the cells express CD44.

Preferably, the diseased retina to be treated by the MLBM cellpopulation and methods of the invention includes activated astrocytes.This can be accomplished by early treatment of the eye when there is anassociated gliosis, or by using a laser to stimulate local proliferationof activated astrocytes.

In addition to therapeutic uses, the isolated myeloid-like bone marrowcell populations of the invention are useful as research tools toinvestigate the physiology of vascular development in the eye, and todeliver specific genes to specific locations (e.g., astrocytes) withinthe eye. Such uses provide a valuable tool for investigation of genefunction and potential therapeutic mechanisms.

BRIEF DESCRIPTION OF THE DRAWINGS

In the DRAWINGS:

FIG. 1 depicts schematic diagrams of developing mouse retina. (a)Development of primary plexus. (b) The second phase of retinal vesselformation. GCL, ganglion cell layer; IPL, inner plexus layer; INL, innernuclear layer; OPL, outer plexus layer; ONL, outer nuclear layer; RPE,retinal pigment epithelium; ON, optic nerve; P, periphery. Panel (c)depicts flow cytometric characterization of bone marrow-derived Lin⁺ HSCand Lin⁻ HSC separated cells. Top row: Dot plot distribution ofnon-antibody labeled cells, in which R1 defines the quantifiable-gatedarea of positive PE-staining; R2 indicates GFP-positive; Middle row:Lin⁻ HSC(C57B/6) and Bottom row: Lin⁺ HSC(C57B/6) cells, each cell linelabeled with the PE-conjugated antibodies for Sca-1, c-kit, Flk-1/KDR,CD31. Tie-2 data was obtained from Tie-2-GFP mice. Percentages indicatepercent of positive-labeled cells out of total Lin⁻ HSC or Lin⁺ HSCpopulation.

FIG. 2 depicts engraftment of Lin⁻ HSCs into developing mouse retina.(a) At four days post-injection (P6) intravitreally injected eGFP⁺ Lin⁻HSC cells attach and differentiate on the retina. (b) Lin⁻ HSC(B6.129S7-Gtrosa26 mice, stained with β-gal antibody) establishthemselves ahead of the vasculature stained with collagen IV antibody(asterisk indicates tip of vasculature). (c) Most of Lin⁺ HSC cells(eGFP⁺) at four days post-injection (P6) were unable to differentiate.(d) Mesenteric eGFP⁺ murine EC four days post-injection (P6). (e) Lin⁻HSCs (eGFP⁺) injected into adult mouse eyes. (f) Low magnification ofeGFP⁺ Lin⁻ HSCs (arrows) homing to and differentiating along thepre-existing astrocytic template in the GFAP-GFP transgenic mouse. (g)Higher magnification of association between Lin⁻ cells (eGFP) andunderlying astrocyte (arrows). (h) Non-injected GFAP-GFP transgeniccontrol. (i) Four days post-injection (P6), eGFP⁺ Lin⁻ HSCs migrate toand undergo differentiation in the area of the future deep plexus. Leftfigure captures Lin⁻ HSC activity in a whole mounted retina; rightfigure indicates location of the Lin⁻ cells (arrows) in the retina (topis vitreal side, bottom is scleral side). (j) Double labeling withα-CD31-PE and α-GFP-alexa 488 antibodies. Seven days after injection,the injected Lin⁻ HSCs (eGFP, red) were incorporated into thevasculature (CD31). Arrowheads indicate the incorporated areas. (k)eGFP⁺ Lin⁻ HSC cells form vessels fourteen days post-injection (P17). (land m) Intra-cardiac injection of rhodamine-dextran indicates that thevessels are intact and functional in both the primary (l) and deepplexus (m).

FIG. 3 shows that eGFP⁺ Lin⁻ HSC cells home to the gliosis (indicated byGFAP expressing-astrocytes, far left image) induced by both laser (a)and mechanical (b) induced injury in the adult retina (asteriskindicates injured site). Far right images are a higher magnification,demonstrating the close association of the Lin⁻ HSCs and astrocytes.Calibration bar=20 μM.

FIG. 4 shows that Lin⁻ HSC cells rescue the vasculature of the retinaldegeneration mouse. (a-d) Retinas at 27 days post-injection (P33) withcollagen IV staining; (a) and (b), retinas injected with Lin⁺ HSC cells(Balb/c) showed no difference in vasculature from normal FVB mice; (c)and (d) retinas injected with Lin⁻ HSCs (Balb/c) exhibited a richvascular network analogous to a wild-type mouse; (a) and (c), frozensections of whole retina (top is vitreal side, bottom is scleral side)with DAPI staining; (b) and (d), deep plexus of retinal whole amount;(e) bar graph illustrating the increase in vascularity of the deepvascular plexus formed in the Lin⁻ HSC cell-injected retinas (n=6). Theextent of deep retinal vascularization was quantified by calculating thetotal length of vessels within each image. Average total length ofvessels/high power field (in microns) for Lin⁻ HSC, Lin⁺ HSC or controlretinas were compared. (f) Comparison of the length of deep vascularplexus after injection with Lin⁻ HSC (R, right eye) or Lin⁺ HSC (L, lefteye) cells from rd/rd mouse. The results of six independent mice areshown (each color represents a separate mouse). (g) and (h) Lin⁻ HSCcells also (Balb/c) rescued the rd/rd vasculature when injected into P15eyes. The intermediate and deep vascular plexus of Lin⁻ HSC (G) or Lin⁺HSC(H) cell injected retinas (one month after injection) are shown.

FIG. 5 depicts photomicrographs of mouse retinal tissue: (a) deep layerof retinal whole mount (rd/rd mouse), five days post-injection (P11)with eGFP⁺ Lin⁻ HSCs visible (gray). (b) and (c) P60 retinal vasculatureof Tie-2-GFP (rd/rd) mice that received Balb/c Lin⁻ cells (b) or Lin⁺HSC cell (c) injection at P6. Only endogenous endothelial cells(GFP-stained) are visible in the left panels of (b) and (c). The middlepanels of (b) and (c) are stained with CD31 antibody; arrows indicatethe vessels stained with CD31 but not with GFP, the right panels of (b)and (c) show staining with both GFP and CD31. (d) α-SMA staining of Lin⁻HSC injected (left panel) and control retina (right panel).

FIG. 6 shows that T2-TrpRS-transfected Lin⁻ HSCs inhibit the developmentof mouse retinal vasculature. (a) Schematic representation of humanTrpRS, T2-TrpRS and T2-TrpRS with an Igk signal sequence at the aminoterminus. (b) T2-TrpRS transfected Lin⁻ HSC-injected retinas expressT2-TrpRS protein in vivo. (1) Recombinant T2-TrpRS produced in E. coli;(2) Recombinant T2-TrpRS produced in E. coli; (3) Recombinant T2-TrpRSproduced in E. coli; (4) control retina; (5) Lin⁻ HSC+pSecTag2A (vectoronly) injected retina; (6) Lin⁻ HSC+pKLe135 (Igk-T2-TrpRS in pSecTag)injected retina. (a) Endogenous TrpRS. (b) Recombinant T2-TrpRS. (c)T2-TrpRS of Lin⁻ HSC injected retina. (c-f) Representative primary(superficial) and secondary (deep) plexuses of injected retinas, sevendays post-injection; (c) and (d) Eyes injected with emptyplasmid-transfected Lin⁻ HSC developed normally; (e) and (f) themajority of T2-TrpRS-transfected Lin⁻ HSC injected eyes exhibitedinhibition of deep plexus; (c) and (e) primary (superficial) plexus; (d)and (f) secondary (deep) plexus). Faint outline of vessels observed in(f) are “bleed-through” images of primary network vessels shown in (e).

FIG. 7 shows the DNA sequence encoding His₆-tagged T2-TrpRS, SEQ ID NO:1.

FIG. 8 shows the amino acid sequence of His₆-tagged T2-TrpRS, SEQ ID NO:2.

FIG. 9 illustrates photomicrographs and electroretinograms (ERG) ofretinas from mice whose eyes were injected with the Lin⁻ HSC and withLin⁺ HSC (controls).

FIG. 10 depicts statistical plots showing a correlation between neuronalrescue (y-axis) and vascular rescue (x-axis) for both the intermediate(Int.) and deep vascular layers of rd/rd mouse eyes treated with Lin⁻HSC.

FIG. 11 depicts statistical plots showing no correlation betweenneuronal rescue (y-axis) and vascular rescue (x-axis) for rd/rd mouseeyes that were treated with Lin⁺ HSC.

FIG. 12 is a bar graph of vascular length (y-axis) in arbitrary relativeunits for rd/rd mouse eyes treated with the Lin⁻ HSC (dark bars) anduntreated (light bars) rd/rd mouse eyes at time points of 1 month (1M),2 months (2M), and 6 months (6M) post-injection.

FIG. 13 includes three bar graphs of the number of nuclei in the outerneural layer (ONR) of rd/rd mice at 1 month (1M), 2 months (2M) and 6months (6M), post-injection, and demonstrates a significant increase inthe number of nuclei for eyes treated with Lin⁻ HSC (dark bars) relativeto control eyes treated with Lin⁺ HSC (light bars).

FIG. 14 depicts plots of the number of nuclei in the outer neural layerfor individual rd/rd mice, comparing the right eye (R, treated with Lin⁻HSC) relative to the left eye (L, control eye treated with Lin⁺ HSC) attime points (post injection) of 1 month (1M), 2 months (2M), and 6months (6M); each line in a given plot compares the eyes of anindividual mouse.

FIG. 15 depicts retinal vasculature and neural cell changes in rd1/rd1(C3H/HeJ, left panels) or wild type mice (C57BL/6, right panels).Retinal vasculature of intermediate (upper panels) or deep (middlepanels) vascular plexuses in whole-mounted retinas (red: collagen IV,green: CD31) and sections (red: DAPI, green: CD31, lower panels) of thesame retinas are shown (P: postnatal day). (GCL: ganglion cell layer,INL: inter nuclear layer, ONL: outer nuclear layer).

FIG. 16 shows that Lin⁻ HSC injection rescues the degeneration of neuralcells in rd1/rd1 mice. (A, B and C), retinal vasculature of intermediate(Int.) or deep plexus and sections of Lin⁻ HSC injected eye (rightpanels) and contralateral control cell (CD31⁻) injected eye (leftpanels) at P30 (A), P60 (B), and P180 (C). (D), the average total lengthof vasculature (+ or − standard error of the mean) in Lin⁻ HSC injectedor control cell (CD31⁻) injected retinas at P30 (left, n=10), P60(middle, n=10), and P180 (right, n=6). Data of intermediate (Int.) anddeep vascular plexus are shown separately (Y axis: relative length ofvasculature). (E), the average numbers of cell nuclei in the ONL at P30(left, n=10), P60 (middle, n=10), or P180 (right, n=6) of control cell(CD31⁻) or Lin⁻ HSC injected retinas (Y axis: relative number of cellnuclei in the ONL). (F), Linear correlations between the length ofvasculature (X axis) and the number of cell nuclei in the ONL (Y axis)at P30 (left), P60 (middle), and P180 (right) of Lin⁻ HSC or controlcell injected retinas.

FIG. 17 demonstrates that retinal function is rescued by Lin⁻ HSCinjection. Electroretinographic (ERG) recordings were used to measurethe function of Lin⁻ HSC or control cell (CD31⁻) injected retinas. (Aand B), Representative cases of rescued and non-rescued retinas 2 monthsafter injection. Retinal section of Lin⁻ HSC injected right eye (A) andCD31⁻ control cell injected left eye (B) of the same animal are shown(green: CD31 stained vasculature, red: DAPI stained nuclei). (C), ERGresults from the same animal shown in (A) and (B).

FIG. 18 shows that a population of human bone marrow cells can rescuedegenerating retinas in the rd1 mouse (A-C). The rescue is also observedin another model of retinal degeneration, rd10 (D-K). A, human Lin⁻ HSCs(hLin⁻ HSCs) labeled with green dye can differentiate into retinalvascular cells after intravitreal injection into C3SnSmn.CB17-Prkdc SCIDmice. (B and C), Retinal vasculature (left panels; upper: intermediateplexus, lower: deep plexus) and neural cells (right panel) in hLin⁻ HSCinjected eye (B) or contralateral control eye (C) 1.5 months afterinjection. (D-K), Rescue of rd10 mice by Lin⁻ HSCs (injected at P6).Representative retinas at P21 (D: Lin⁻ HSCs, H: control cells), P30 (E:Lin⁻ HSCs, I: control cells), P60 (F: Lin⁻ HSCs, J: control cells), andP105 (G: Lin⁻ HSCs, K: control cells) are shown (treated and controleyes are from the same animal at each time point). Retinal vasculature(upper image in each panel is the intermediate plexus; the middle imagein each panel is the deep plexus) was stained with CD31 (green) andCollagen IV (red). The lower image in each panel shows a cross sectionmade from the same retina (red: DAPI, green: CD31).

FIG. 19 demonstrates that crystallin αA is up regulated in rescued outernuclear layer cells after treatment with Lin⁻ HSCs but not incontralateral eyes treated with control cells. Left panel; IgG controlin rescued retina, Middle panel; crystallin αA in rescued retina, Rightpanel; crystallin αA in non-rescued retina.

FIG. 20 includes tables of genes that are upregulated in murine retinasthat have been treated with the Lin⁻ HSCs of the present invention. (A)Genes whose expression is increased 3-fold in mouse retinas treated withmurine Lin⁻ HSCs. (B) Crystallin genes that are upregulated in mouseretinas treated with murine Lin⁻ HSC. (C) Genes whose expression isincreased 2-fold in mouse retinas treated with human Lin⁻ HSCs. (D)Genes for neurotrophic factors or growth factors whose expression isupregulated in mouse retinas treated with human Lin⁻ HSCs.

FIG. 21 illustrates the distribution of CD31 and integrin α6 surfaceantigens on CD133 positive (DC133⁺) and CD133 negative (CD133⁻) humanLin⁻ HSC populations. The left panels show flow cytometry scatter plots.The center and right panels are histograms showing the level of specificantibody expression on the cell population. The Y axis represents thenumber of events and the X axis shows the intensity of the signal. Afilled histogram shifted to the right of the outlined (control)histogram represents an increased fluorescent signal and expression ofthe antibody above background level.

FIG. 22 illustrates postnatal retinal development in wild-type C57/B16mice raised in normal oxygen levels (normoxia), at post natal days P0through P30.

FIG. 23 illustrates oxygen-induced retinopathy model in C57/B16 miceraised in high oxygen levels (hyperoxia; 75% oxygen) between P7 and P12,followed by normoxia from P12-P17.

FIG. 24 demonstrates vascular rescue by treatment with the Lin⁻ HSCpopulations in the oxygen-induced retinopathy (OIR) model.

FIG. 25 shows rescued photoreceptors in rd1 mouse outer nuclear layer(ONL) following intravitreal injection of Lin-HSC are predominantlycones. A small percentage of photoreceptors in the wild type mouseretina (upper panel) were cones as evidenced by expression of red/greencone opsin (A) while most cells of the ONL were positive for rodspecific rhodopsin (B). Retinal vasculature autofluoresces withpre-immune serum (C) but nuclear layers were completely negative forstaining with rod or cone-specific opsins. Rd/rd mouse retinas (lowerpanels) had a diminished inner nuclear layer and a nearly completelyatrophic ONL, both of which were negative for cone (D) or rod (Panel G)opsin. Control, CD31− HSC treated eyes are identical to non-injectedrd/rd retinas, without any staining for cone (E) or rod (H) opsin.Lin-HSC treated contralateral eyes exhibited a markedly reduced, butclearly present ONL that is predominantly comprised of cones, asevidenced by positive immunoreactivity for cone red/green opsin (F). Asmall number of rods were also observed (I).

FIG. 26 shows scatter plots from flow cytometry characterization oflineage negative and lineage positive stem cell populations (upper leftand lower left plots, respectively) showing percentages of cells thatexpress the CD44 antigen (data points in red); as well as plots of CD31negative and CD31 positive cell populations (upper right and lower rightplots, respectively), showing percentages of cells that express the CD44antigen (data points in red).

FIG. 27 shows scatter plots from flow cytometry characterization of alineage negative cell population that expresses a significant level ofCD44 antigen (left set of plots) and a sub-population of bone marrowcells that do not express a significant level of CD44 antigen (right setof plots) illustrating the relative percentages of cells expressingvarious other cell surface antigens.

FIG. 28 shows photomicrographic images of a retina from a mouseintravitreally injected with cells from the MLBM cell population of theinvention (left panel) compared to a retina from a mouse intravitreallyinjected with CD44^(lo) cells.

FIG. 29 shows photomicrographic images of retinas from eyes injectedwith cells from the MLBM cell population (CD44^(hi)) and with CD44^(lo)cells.

FIG. 30 shows bar graphs demonstrating the beneficial effects of theMLBM cell population for ameliorating pathogenic angiogensis andpromoting beneficial physiological revascularization of mouse retinas inthe oxygen induced retinopathy model of retinopathy of prematurity. Theupper graph compares pre-retinal neovascular tuft area for controlretina (first bar), retina treated with CD44^(lo) cells (middle bar) andretinas treated with cells from the MLBM cell population (right bar).The lower graph compares vascular obliteration area for control retina(first bar), retina treated with CD44^(lo) cells (middle bar) andretinas treated with cells from the MLBM cell population (right bar).

FIG. 31 is a photomicrographic image demonstrating that once cells fromthe MLBM cell population have incorporated into the vasculature of theretina, the cells express vascular endothelial growth factor (VEGF), asindicated by the green staining of the cells in the lower portion of theimage.

FIG. 32 depicts photomicrographic images demonstrating that cells fromthe CD11b⁺ MLBM cell population of the invention selectively target thevasculature of the retina.

FIG. 33 depicts photomicrographic images demonstrating that CD44⁻ CD11b⁻bone marrow cells do not selectively target the vasculature of theretina.

FIG. 34 shows the amino acid residue sequence of the T2 fragment ofTrpRS (SEQ ID NO: 3) and of the T2-TrpRS-GD variation thereof (SEQ IDNO: 4).

FIG. 35 shows the amino acid residue sequence of mini-TrpRS (SEQ ID NO:5).

FIG. 36 shows the amino acid residue sequence of T1-TrpRS (SEQ ID NO:6).

FIG. 37 shows normal retinal vascular development in the mouse, theoxygen-induced retinopathy (OIR) model, and the rescue effect followingintra-vitreal transplantation of Lin− bone-marrow derived-cells. Themouse is born with a largely avascular retina. as shown at postnatal day2 (P2) (Panel a, retinal whole-mount) where the vessels are found in thesuperficial retina occupying a single plane as shown in b. Panels b,dand f are images taken from 3D renderings of en face confocal z-seriesdata sets rotated 90 degrees. During the first week after birth, thesuperficial retinal vasculature grows in a radial fashion from the opticnerve head nearly reaching the periphery by P10 (c). The deep retinalvasculature is then established from branching of the superficial layerduring the second week (d). Finally, a third plexus of vessels formsbetween the first two, and establishes the mature retinal vasculature ataround P30 (e,f). Panel g shows that exposure to hyperoxia in the OIRmodel causes central vaso-obliteration as shown here at P10. Panel hshows that after removal to normoxia at P12, the central retina startsto revascularize and characteristic pre-retinal neovascular tufts areformed at the interface between the vascularized (peripheral) andavascular (central) retina. These tufts stain strongly with isolectin.Panels i-n show that Lin⁻ hematopoietic progenitor cells promotevascular repair in the OIR model. Lin⁻ cells injected intravitreallyprior to high oxygen exposure dramatically accelerate revascularizationof the central retina when compared to the vehicle-treated fellow eye atP17. While retinas treated with vehicle show partial absence of thesuperficial vasculature (i) and complete absence of the deep retinalvasculature (k,m), the Lin⁻ cell-treated fellow eye shows relativelynormal retinal vasculature (j) with all three plexuses present (k,m).Panel o shows that at P17, OIR eyes treated with Lin⁻ cells are fullyrevascularized significantly more often than uninjected eyes or thoseinjected with vehicle. Vessels were visualized by cardiac perfusion offluorescein-dextran, as shown in Panels a-f,i,j and by GS lectin inPanels g,h,k-n. Nuclei in Panels k-n were labeled with DAPI.

FIG. 38 shows Lin⁻ cells accelerate retinal revascularization and reducepre-retinal neovascular tuft formation in OIR. Panels a-d show acomputer image analysis method was used to calculate the area of retinalvessel obliteration, as well as pre-retinal neovascular tuft formation(red) in retinal whole-mounts from OIR eyes at postnatal day 17. Panel eshows retinas treated with Lin− cells prior to hyperoxia showed analmost 6-fold reduction in obliterated area versus uninjected controlsand an approximately 5-fold reduction compared to eyes treated withvehicle alone. Panel f shows Lin⁻ cell treatment significantly reducedtwo-dimensional area of neovascular tufts compared to uninjected eyesand vehicle-treated eyes. Panel g shows Lin⁻ cell-transplantation iseffective at reducing the area of obliteration not only whenadministered prior to hyperoxia, but also at P9-P12 during hyperoxia andjust after return to normoxia. (graphs represent Mean±SEM; *p<0.001).

FIG. 39 shows bone marrow cell treatment has little or no long termtoxic effects. Retinas evaluated at 5 or 6 months after receiving Lin⁻cell treatment have normal-appearing retinal vasculature and the neuralretina appears histologically preserved on cross sections (a-f,non-injected versus Lin⁻ cell-injected retina 6 months post-transplant).No tumors were observed, and the only abnormality was an occasional“rosette” in the neural retina which could also be seen in controlnon-injected eyes (g,h).

FIG. 40 shows CD44^(HI) cells are prevalent in the Lin⁻ population andeffectively promote vascular repair in the OIR model. Panel a shows bonemarrow contains CD44^(HI) and CD44^(LO) fractions and the Lin⁻population is enriched for CD44^(HI) cells compared to control CD cells.Insets show light scattering properties of the CD44^(HI) cells which aretypical of monocytes and granulocytes, while light-scattering propertiesof CD44^(LO) cells are typical of lymphocytes. Panel b showsrepresentative P17 retinas from eyes treated with CD44^(LO) andCD44^(HI) bone marrow cells prior to oxygen exposure. The lower panelsexemplify the quantified areas of obliteration and neovascularization atP17 used to create the data shown in panel c. Panel c shows vascularobliteration and pre-retinal neovascularization are reduced in eyestreated with CD44^(HI) cells with efficacy similar to eyes treated withLin⁻ cells. Areas of vascular obliteration (*) and pre-retinalneovascularization (**) were significantly lower in CD44^(HI) and Lin⁻eyes compared with vehicle injection or no injection (p<10⁻⁵ in allcases). Area of obliteration in Lin⁻ cell-treated eyes was also reducedcompared to CD44^(HI) (p=0.03), but to a much lesser degree. Areas ofpre-retinal neovascularization did not significantly differ between Lin⁻and CD44^(HI)-treated eyes (p=0.25).

FIG. 41 shows the CD44^(HI) subpopulation expresses myeloid markers. InPanel a, two-color flow cytometry was used to further characterize CD44populations. All cells were labeled with an antibody against CD44 andco-labeled with the various antibodies shown. The CD44^(HI) populationshowed strong labeling for CD11a, CD11b and Ly6GC. Fractions of CD44hicells were positive for CD14, F4/80, cKit, and CD115. Most of theseantigens are present on myeloid lineage cells. CD44lo cells labeledstrongly with Ter119 and CD45R B220, which are markers for erythroblastsand B cells, respectively.

FIG. 42 shows that CD44^(HI) cells take on a perivascular localizationin the retina. Confocal imaging was used to create a series of images inthe z dimension which were then rendered into 3D. In Panel a, aprojection of this is shown the CD31-labeled vascular endothelium andGFP expression from the introduced bone marrow cells are shown. The bonemarrow cell appears to have assumed a perivascular position. 3D datashow that the lumen of the vessel and the relative position of the GFP⁺bone marrow cell are visualized. The numbers listed in (b) correspond tocross-sectional positions indicated in (a). The GFP signal was detectedoutside of the lumen in all cases, except Panel b, No. 3, which was asection through the cell body with intense fluorescence wherebleed-through of the signal was evident.

FIG. 43 shows an in situ analysis of injected CD44^(HI) bone marrowcells in the OIR model. Labeling of a control retina that received nocell treatment shows the presence of endogenous F4/80+ perivascularcells (a-c). Injected CD44^(HI) cells target the retinal vasculature andhave a localization, morphology and F4/80 expression pattern similar toendogenous cells (d-i). Transplanted perivascular bone marrow cells loseCD44 expression, while cells not associated with the retinal vasculatureretain CD44 expression (j-o).

FIG. 44 shows an expression array analysis, which revealed a highexpression of myeloid-associated genes in the CD44^(HI) population whilethe CD44^(LO) cells expressed genes associated with lymphoid cells.AFFYMETRIX® arrays were used to compare gene expression profiles betweenthese two bone marrow cell populations. Genes shown had a minimum 5-folddifference in expression. A significantly higher level of CD44expression in the CD44^(HI) population was observed versus CD44^(LO)cells.

FIG. 45 demonstrates that CD44^(HI) cells can differentiate into cellswith microglial characteristics. Panels A and B show that injectedCD44^(HI) cells express CD11b and F4/80 and have morphology andperivascular localization similar to endogenous microglia. Panel Cprovides 3d imaging of the perivascular localization of an injectedCD44^(HI) cell. Panel D shows a high magnification view of themorphology of injected CD44^(HI) cells.

FIG. 46 demonstrates that CD44^(HI) cells can be isolated by negativeselection. Panel A shows that depletion of mouse bone marrow by MACSusing antibodies selective for CD45R/B220, TER119, and CD3e yields apopulation of cells that are greater than 90 percent CD44^(HI) cells.Panel B shows the negative fraction (CD44^(HI) population) isessentially free from CD45R/B220, TER119, and CD3e cells. Panel C showsnegatively selected CD44^(HI) cells retain retinal targeting anddifferentiation capabilities.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Bone marrow cells include a sub-population of cells that express theCD44 antigen (i.e., the hyaluronic acid receptor) and CD11b (integrinαM). A myeloid-like population of bone marrow cells enriched in CD44 andCD11b expressing cells can be isolated from bone marrow by treating bonemarrow cells with an antibody to CD44 antigen (anti-CD44) and/or anantibody to CD11b antigen (anti-CD11b), and then selecting cells thatimmunoreact with the antibody. The antibody then can be removed from thecells by methods that are well known in the art. The cells can beselected, for example, using by flow cytometry, using antibodies boundto or coated on beads followed by filtration, or other separationmethods that are well known in the art. A majority of the selected cellsare lineage negative and express both the CD44 antigen and the CD11bantigen, regardless of which antibody is utilized in the isolation.

Bone marrow includes stem cells. Stem cells are typically identified bythe distribution of antigens on the surface of the cells (for a detaileddiscussion see Stem Cells: Scientific Progress and Future Directions, areport prepared by the National Institutes of Health, Office of SciencePolicy, June 2001, Appendix E: Stem Cell Markers, which is incorporatedherein by reference to the extent pertinent). Approximately 75% oflineage negative hematopoietic stems cells isolated from bone marrow arealso CD44 positive. In a preferred embodiment, a majority of the cellsfrom the MLBM cell population are lineage negative hematopoietic stemcells (i.e., CD44⁺ Lin⁻ HSC).

The present invention provides a method of ameliorating vascular andneuronal degeneration in the retina of a mammal that suffers from anocular disease. Isolated MLBM cell population of the invention isadministered to the retina of the mammal, preferably by intravitrealinjection. The cells are administered in an amount sufficient toameliorate vascular and/or neuronal degeneration in the retina.Preferably, the isolated MLBM cell population is autologous to themammal to be treated. Preferably, the cells from the MLBM cellpopulation are administered in a physiologically tolerable medium, suchas phosphate buffered saline (PBS).

A preferred method comprises isolating the MLBM cell population from thebone marrow of the mammal to be treated and then administering the cellsto the mammal in a number sufficient to ameliorate the vascular and/orneuronal degeneration of the retina. The cells can be isolated from amammal suffering from an ocular degenerative disease, preferably at anearly stage of the ocular disease or from a healthy mammal known to bepredisposed to an ocular degenerative disease (i.e., through geneticpredisposition). In the latter case, the isolated MLBM cell populationcan be stored after isolation, and can then be injected prophylacticallyduring early stages of a later developed ocular disease. Preferably thediseased retina includes activated astrocytes, to which the cells fromthe MLBM cell population are targeted. Accordingly, early treatment ofthe eye when there is an associated gliosis is beneficial.Alternatively, the retina can be treated with a laser to stimulate localproliferation of activated astrocytes in the retina prior toadministering the autologous MLBM cell population.

Hematopoietic stem cells are stem cells that are capable of developinginto various blood cell types e.g., B cells, T cells, granulocytes,platelets, and erythrocytes. The lineage surface antigens are a group ofcell-surface proteins that are markers of mature blood cell lineages,including CD2, CD3, CD11, CD11a, Mac-1 (CD11b:CD18), CD14, CD16, CD19,CD24, CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119,CD56, CD64, CD68, CD86 (B7.2), CD66b, human leucocyte antigen DR(HLA-DR), and CD235a (Glycophorin A). Hematopoietic stem cells that donot express significant levels of these antigens are commonly referredto a lineage negative (Lin⁻). Human hematopoietic stem cells commonlyexpress other surface antigens such as CD31, CD34, CD117 (c-kit) and/orCD133. Murine hematopoietic stem cells commonly express other surfaceantigens such as CD34, CD117 (c-kit), Thy-1, and/or Sca-1.

Isolated hematopoietic stem cells that do not express significant levelsof a “lineage surface antigen” (Lin) on their cell surfaces are referredto herein as “lineage negative” or “Lin” hematopoietic stem cells i.e.,Lin⁻ HSC. A majority of the cells of the MLBM cell populations of thepresent invention are Lin⁻ and express both a relatively high amount ofthe CD44 antigen (CD44^(hi)) as well as the CD11b antigen. TheseCD44⁺CD11b⁺Lin⁻ HSC are capable of incorporating into developingvasculature and then differentiating to become vascular endothelialcells.

As used herein and in the appended claims, the phrase “adult” inreference to bone marrow and bone marrow cells, includes bone marrowisolated postnatally, i.e., from juvenile and adult individuals, asopposed to embryos. Accordingly, the term “adult mammal” refers to bothjuvenile (postnatal) and fully mature mammals, as opposed to an embryoor prenatal individual.

The isolated MLBM cell populations of the present invention selectivelytarget astrocytes and incorporate into the retinal neovasculature whenintravitreally injected into the eye of the mammalian species, such as amouse or a human, from which the cells were isolated.

The isolated MLBM cell populations of the present invention includecells that differentiate to endothelial cells and generate vascularstructures within the retina. In particular, the MLBM cell population ofthe present invention is useful for the treatment of retinal neovascularand retinal vascular degenerative diseases, and for repair of retinalvascular injury. The MLBM cell population of the present invention alsopromotes neuronal rescue in the retina and promote upregulation ofanti-apoptotic genes. Additionally, the MLBM cell population of theinvention can be utilized to treat retinal defects in the eyes ofneonatal mammals, such as mammals suffering from oxygen inducedretinopathy or retinopathy of prematurity.

It has been found that bone marrow cells that do not express CD44(CD44^(LO) cells) generally express one or more of the following cellmarkers: Ter119, CD45RB220, and CD3e. Utilizing this fact, CD44^(HI)MLBM cells of the present invention can be isolated by a methodinvolving negative cell-marker selection. The method comprisescontacting a plurality of bone marrow cells with antibodies specific forTer119, CD45RB220, and CD3e, removing cells from the plurality of bonemarrow cells that immunoreact with Ter119, CD45RB220, and CD3eantibodies, and recovering myeloid-like bone marrow cells that aredeleted in Ter119, CD45RB220, and CD3e-expressing cells. Using thismethod, a cell population can be recovered in which greater than 90percent of the cells express CD44.

The present invention also provides a method of treating ocular diseasesin a mammal comprising isolating from the bone marrow of the mammal aMLBM cell population, and intravitreally injecting cells from the MLBMcell population into an eye of the mammal in a number sufficient toarrest the disease. The present method can be utilized to treat oculardiseases such as retinal degenerative diseases, retinal vasculardegenerative diseases, ischemic retinopathies, vascular hemorrhages,vascular leakage, and choroidopathies in neonatal, juvenile or fullymature mammals. Examples of such diseases include age related maculardegeneration (ARMD), diabetic retinopathy (DR), presumed ocularhistoplasmosis (POHS), retinopathy of prematurity (ROP), sickle cellanemia, and retinitis pigmentosa, as well as retinal injuries.

The number of cells from the MLBM cell population injected into the eyeis sufficient for arresting the disease state of the eye. For example,the amount of injected cells can be effective for repairing retinaldamage of the eye, stabilizing retinal neovasculature, maturing retinalneovasculature, and preventing or repairing vascular leakage andvascular hemorrhage.

Cells from the MLBM cell population of the present invention can betransfected with therapeutically useful genes, such as genes encodingantiangiogenic proteins for use in ocular, cell-based gene therapy andgenes encoding neurotrophic agents to enhance neuronal rescue effects.

The transfected cells can include any gene which is therapeuticallyuseful for treatment of retinal disorders. In one preferred embodiment,the transfected cells from the MLBM cell population of the presentinvention include a gene operably encoding an antiangiogenic peptide,including proteins, or protein fragments such as TrpRS or antiangiogenic(i.e., angiostatic) fragments thereof, e.g., the fragments of TrpRSdesignated T2-TrpRS (SEQ ID NO: 3 in FIG. 34), T2-TrpRS-GD (SEQ ID NO: 4in FIG. 34), both of which are preferred angiostatic peptides, as wellas mini-TrpRS (SEQ ID NO: 5 in FIG. 35), and T1-TrpRS(SEQ ID NO: 6 inFIG. 36). The transfected cells from the MLBM cell population encodingan antiangiogenic peptide of the present invention are useful fortreatment of retinal diseases involving abnormal vascular development,such as diabetic retinopathy, and like diseases. Preferably, the cellsfrom the MLBM cell population are human cells.

In another preferred embodiment, the transfected cells from the MLBMcell population of the present invention include a gene operablyencoding a neurotrophic agent such as nerve growth factor,neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophicfactor, retinal pigmented epithelium-derived neurotrophic factor,insulin-like growth factor, glial cell line-derived neurotrophic factor,brain-derived neurotrophic factor, and the like. Such neurotrophic cellsfrom the MLBM cell population are useful for promoting neuronal rescuein retinal neuronal degenerative diseases such as glaucoma and retinitispigmentosa, in treatment of injuries to the retinal nerves, and thelike. Implants of ciliary neurotrophic factor have been reported asuseful for the treatment of retinitis pigmentosa (see Kirby et al. 2001,Mol. Ther. 3(2):241-8; Farrar et al. 2002, EMBO Journal 21:857-864).Brain-derived neurotrophic factor reportedly modulates growth associatedgenes in injured retinal ganglia (see Fournier, et al., 1997, J.Neurosci. Res. 47:561-572). Glial cell line derived neurotrophic factorreportedly delays photoreceptor degeneration in retinitis pigmentosa(see McGee et al. 2001, Mol. Ther. 4(6):622-9).

The present invention also provides methods for treating ocularangiogenic diseases by administering transfected cells from the MLBMcell population of the present invention by intravitreal injection ofthe cells into the eye. Such transfected cells from the MLBM cellpopulation comprise cells from the MLBM cell population transfected witha therapeutically useful gene, such as a gene encoding antiangiogenic orneurotrophic gene product. Preferably the transfected cells from theMLBM cell population are human cells.

Preferably, at least about 1×10⁵ cells from the MLBM cell population ortransfected cells from the MLBM cell population are administered byintravitreal injection to a mammalian eye suffering from a retinaldegenerative disease. The number of cells to be injected may depend uponthe severity of the retinal degeneration, the age of the mammal andother factors that will be readily apparent to one of ordinary skill inthe art of treating retinal diseases. The cells from the MLBM cellpopulation may be administered in a single dose or by multiple doseadministration over a period of time, as determined by the clinician incharge of the treatment.

The MLBM cell populations of the present invention is useful for thetreatment of retinal injuries and retinal defects involving aninterruption in or degradation of the retinal vasculature or retinalneuronal degeneration. Human MLBM cell populations also can be used togenerate a line of genetically identical cells, i.e., clones, for use inregenerative or reparative treatment of retinal vasculature, as well asfor treatment or amelioration of retinal neuronal degeneration. Furthermore, the MLBM cell populations of the present invention are useful asresearch tools to study retinal vascular development and to delivergenes to selected cell targets, such as astrocytes.

Murine Retinal Vascular Development.

A Model for Ocular Angiogenesis. The mouse eye provides a recognizedmodel for the study of mammalian retinal vascular development, such ashuman retinal vascular development. During development of the murineretinal vasculature, ischemia-driven retinal blood vessels develop inclose association with astrocytes. These glial elements migrate onto thethird trimester human fetus, or the neonatal rodent, retina from theoptic disc along the ganglion cell layer and spread radially. As themurine retinal vasculature develops, endothelial cells utilize thisalready established astrocytic template to determine the retinalvascular pattern (See FIG. 1 (a and b)). FIG. 1 (a and b) depictsschematic diagrams of developing mouse retina. Panel (a) depictsdevelopment of the primary plexus (dark lines at upper left of thediagram) superimposed over the astrocyte template (light lines) whereas,(b) depicts the second phase of retinal vessel formation. In FIG. 1, GCLstands for ganglion cell layer; IPL stands for inner plexus layer; INLstands for inner nuclear layer; OPL stands for outer plexus layer; ONLstands for outer nuclear layer; RPE stands for retinal pigmentepithelium; ON stands for optic nerve; and P stands for periphery.

At birth, retinal vasculature is virtually absent. By postnatal day 14(P14) the retina has developed complex primary (superficial) andsecondary (deep) layers of retinal vessels coincident with the onset ofvision. Initially, spoke-like peripapillary vessels grow radially overthe pre-existing astrocytic network towards the periphery, becomingprogressively interconnected by capillary plexus formation. Thesevessels grow as a monolayer within the nerve fiber through P10 (FIG. 1(a)). Between P7-P8 collateral branches begin to sprout from thisprimary plexus and penetrate into the retina to the outer plexiformlayer where they form the secondary, or deep, retinal plexus. By P21,the entire network undergoes extensive remodeling and a tertiary, orintermediate, plexus forms at the inner surface of inner nuclear layer(FIG. 1 (b)).

The neonatal mouse retinal angiogenesis model is useful for studying therole of HSC during ocular angiogenesis for several reasons. In thisphysiologically relevant model, a large astrocytic template exists priorto the appearance of endogenous blood vessels, permitting an evaluationof the role for cell-cell targeting during a neovascular process. Inaddition, this consistent and reproducible neonatal retinal vascularprocess is known to be hypoxia-driven, in this respect havingsimilarities to many retinal diseases in which ischemia is known to playa role.

Enrichment of Endothelial Progenitor Cells (EPC) From Bone Marrow.

Although cell surface marker expression has been extensively evaluatedon the EPC population found in preparations of HSC, markers thatuniquely identify EPC are still poorly defined. To enrich for EPC,hematopoietic lineage marker positive cells (Lin⁺), i.e., B lymphocytes(CD45), T lymphocytes (CD3), granulocytes (Ly-6G), monocytes (CD11), anderythrocytes (TER-119), were depleted from bone marrow mononuclear cellsof mice. Sca-1 antigen was used to further enrich for EPC. A comparisonof results obtained after intravitreal injection of identical numbers ofeither Lin⁻ Sca-1⁺ cells or Lin⁻ cells, no difference was detectedbetween the two groups. In fact, when only Lin⁻ Sca-1⁻ cells wereinjected, far greater incorporation into developing blood vessels wasobserved.

Lin⁻ HSC populations are enriched with EPCs, based on functional assays.Furthermore, Lin⁺ HSC populations functionally behave quite differentlyfrom the Lin⁻ HSC populations. Epitopes commonly used to identify EPCfor each fraction (based on previously reported in vitrocharacterization studies) were also evaluated. While none of thesemarkers were exclusively associated with the Lin⁻ fraction, all wereincreased about 70 to about 1800% in the Lin⁻ HSC, compared to the Lin⁺HSC fraction (FIG. 1 (c)). FIG. 1, Panel (c) illustrates flow cytometriccharacterization of bone marrow-derived Lin⁺ HSC and Lin⁻ HSC separatedcells. The top row of Panel (c) shows a hematopoietic stem cell dot plotdistribution of non-antibody labeled cells. R1 defines thequantifiable-gated area of positive PE-staining; R2 indicatesGFP-positive. Dot plots of Lin⁻ HSC are shown in the middle row and dotplots of Lin⁺ HSC are shown in the bottom row. The C57B/6 cells werelabeled with the PE-conjugated antibodies for Sca-1, c-kit, Flk-1/KDR,CD31. Tie-2 data was obtained from Tie-2-GFP mice. The percentages inthe corners of the dot plots indicate the percent of positive-labeledcells out of total Lin⁻ or Lin⁺ HSC population. Interestingly, acceptedEPC markers like Flk-1/KDR, Tie-2, and Sca-1 were poorly expressed and,thus, not used for further fractionation.

Lin⁻ HSC can be isolated by (a) extracting bone marrow from an adultmammal; (b) separating a plurality of monocytes from the bone marrow;(c) labeling the monocytes with biotin-conjugated lineage panelantibodies to one or more lineage surface antigens, preferably lineagesurface antigens selected from the group consisting of CD2, CD3, CD4,CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45,Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2),CD66b, human leucocyte antigen DR (HLA-DR), and CD235a (Glycophorin A);(d) removing monocytes that are positive for said one or more lineagesurface antigens from the plurality of monocytes; and (e) recovering apopulation of lineage negative hematopoietic stem cells therefrom.

When the Lin⁻ HSC are isolated from adult human bone marrow, preferablythe monocytes are labeled with biotin-conjugated lineage panelantibodies to lineage surface antigens CD2, CD3, CD4, CD11a, Mac-1,CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 (B7.2), andCD235a. When the Lin⁻ HSC are isolated from adult murine bone marrow,preferably the monocytes are labeled with biotin-conjugated lineagepanel antibodies to lineage surface antigens CD3, CD11, CD45, Ly-6G, andTER-119.

Intravitreally Injected HSC Lin⁻ Cells Contain EPC That TargetAstrocytes and Incorporate into Developing Retinal Vasculature.

To determine whether intravitreally injected Lin⁻ HSC can targetspecific cell types of the retina, utilize the astrocytic template andparticipate in retinal angiogenesis, approximately 10⁵ cells from a Lin⁻HSC composition of the present invention or Lin⁺ HSC cells (control,about 10⁵ cells) isolated from the bone marrow of adult (GFP or LacZtransgenic) mice were injected into postnatal day 2 (P2) mouse eyes.Four days after injection (P6), many cells from the Lin⁻ HSC compositionof the present invention, derived from GFP or LacZ transgenic mice wereadherent to the retina and had the characteristic elongated appearanceof endothelial cells (FIG. 2 (a)). FIG. 2 illustrates engraftment ofLin⁻ cells into developing mouse retina. As shown in FIG. 2, Panel (a),the four days post-injection (P6) intravitreally injected eGFP+ Lin⁻ HSCattach and differentiate on the retina.

In many areas of the retinas, the GFP-expressing cells were arranged ina pattern conforming to underlying astrocytes and resembled bloodvessels. These fluorescent cells were observed ahead of the endogenous,developing vascular network (FIG. 2 (b)). Conversely, only a smallnumber of Lin⁺ HSC (FIG. 2 (c)), or adult mouse mesenteric endothelialcells (FIG. 2 (d)) attached to the retinal surface. In order todetermine whether cells from an injected Lin⁻ HSC population could alsoattach to retinas with already established vessels, a Lin⁻ HSCcomposition was injected into adult eyes. Interestingly, no cells wereobserved to attach to the retina or incorporate into established, normalretinal blood vessels (FIG. 2 (e)). This indicates that the Lin⁻ HSCcompositions of the present invention do not disrupt a normallydeveloped vasculature and will not initiate abnormal vascularization innormally developed retinas.

In order to determine the relationship between an injected Lin⁻ HSCcompositions of the present invention and retinal astrocytes, atransgenic mouse was used, which expressed glial fibrillary acidicprotein (GFAP, a marker of astrocytes) and promoter-driven greenfluorescent protein (GFP). Examination of retinas of these GFAP-GFPtransgenic mice injected with Lin⁻ HSC from eGFP transgenic micedemonstrated co-localization of the injected eGFP EPC and existingastrocytes (FIG. 2 (f-h), arrows). Processes of eGFP+Lin⁻ HSC wereobserved to conform to the underlying astrocytic network (arrows, FIG. 2(g)). Examination of these eyes demonstrated that the injected, labeledcells only attached to astrocytes; in P6 mouse retinas, where theretinal periphery does not yet have endogenous vessels, injected cellswere observed adherent to astrocytes in these not yet vascularizedareas. Surprisingly, injected, labeled cells were observed in the deeperlayers of the retina at the precise location where normal retinalvessels will subsequently develop (FIG. 2 (i), arrows).

To determine whether injected Lin⁻ HSC are stably incorporated into thedeveloping retinal vasculature, retinal vessels at several later timepoints were examined. As early as P9 (seven days after injection), Lin⁻HSC incorporated into CD31⁺ structures (FIG. 2 (j)). By P16 (14 daysafter injection), the cells were already extensively incorporated intoretinal vascular-like structures (FIG. 2 (k)). When rhodamine-dextranwas injected intravascularly (to identify functional retinal bloodvessels) prior to sacrificing the animals, the majority of Lin⁻ HSC werealigned with patent vessels (FIG. 2 (l)). Two patterns of labeled celldistribution were observed: (1) in one pattern, cells were interspersedalong vessels in between unlabeled endothelial cells; and (2) the otherpattern showed that vessels were composed entirely of labeled cells.Injected cells were also incorporated into vessels of the deep vascularplexus (FIG. 2 (m)). While sporadic incorporation of Lin⁻ HSC-derivedEPC into neovasculature has been previously reported, this is the firstreport of vascular networks being entirely composed of these cells. Thisdemonstrates that cells from a population of bone marrow-derived Lin⁻HSC, injected intravitreally, can efficiently incorporate into any layerof the forming retinal vascular plexus.

Histological examination of non-retinal tissues (e.g., brain, liver,heart, lung, bone marrow) did not demonstrate the presence of any GFPpositive cells when examined up to 5 or 10 days after intravitrealinjection. This indicates that a sub-population of cells within the Lin⁻HSC fraction selectively target to retinal astrocytes and stablyincorporate into developing retinal vasculature. Since these cells havemany characteristics of endothelial cells (association with retinalastrocytes, elongate morphology, stable incorporation into patentvessels and not present in extravascular locations), these cellsrepresent EPC present in the Lin⁻ HSC population. The targetedastrocytes are of the same type observed in many of the hypoxicretinopathies. It is well known that glial cells are a prominentcomponent of neovascular fronds of tufts observed in DR and other formsof retinal injury. Under conditions of reactive gliosis andischemia-induced neovascularization, activated astrocytes proliferate,produce cytokines, and up-regulate GFAP, similar to that observed duringneonatal retinal vascular template formation in many mammalian speciesincluding humans.

Lin⁻ HSC populations will target activated astrocytes in adult mouseeyes as they do in neonatal eyes, Lin⁻ HSC cells were injected intoadult eyes with retinas injured by photo-coagulation (FIG. 3 (a)) orneedle tip (FIG. 3 (b)). In both models, a population of cells withprominent GFAP staining was observed only around the injury site (FIG. 3(a and b)). Cells from injected Lin⁻ HSC compositions localized to theinjury site and remained specifically associated with GFAP-positiveastrocytes (FIG. 3 (a and b)). At these sites, Lin⁻ HSC cells were alsoobserved to migrate into the deeper layer of retina at a level similarto that observed during neonatal formation of the deep retinalvasculature. Uninjured portions of retina contained no Lin⁻ HSC cells,identical to that observed when Lin⁻ HSC were injected into normal,uninjured adult retinas (FIG. 2 (e)). These data indicate that Lin⁻ HSCcompositions can selectively target activated glial cells in injuredadult retinas with gliosis as well as neonatal retinas undergoingvascularization.

Intravitreally Injected Lin⁻ HSC Can Rescue and Stabilize DegeneratingVasculature.

Since intravitreally injected Lin⁻ HSC compositions target astrocytesand incorporate into the normal retinal vasculature, these cells alsostabilize degenerating vasculature in ischemic or degenerative retinaldiseases associated with gliosis and vascular degeneration. The rd/rdmouse is a model for retinal degeneration that exhibits profounddegeneration of photoreceptor and retinal vascular layers by one monthafter birth. The retinal vasculature in these mice develops normallyuntil P16 at which time the deeper vascular plexus regresses; in mostmice the deep and intermediate plexuses have nearly completelydegenerated by P30.

To determine whether HSC can rescue the regressing vessels, Lin⁺ or Lin⁻HSC (from Balb/c mice) were injected into rd/rd mice intravitreally atP6. By P33, after injection with Lin⁺ cells, vessels of the deepestretinal layer were nearly completely absent (FIG. 4 (a and b)). Incontrast, most Lin⁻ HSC-injected retinas by P33 had a nearly normalretinal vasculature with three parallel, well-formed vascular layers(FIG. 4 (a and d)). Quantification of this effect demonstrated that theaverage length of vessels in the deep vascular plexus of Lin⁻ injectedrd/rd eyes was nearly three times greater than untreated or Lin⁺cell-treated eyes (FIG. 4 (e)). Surprisingly, injection of a Lin⁻ HSCcomposition derived from rd/rd adult mouse (FVB/N) bone marrow alsorescued degenerating rd/rd neonatal mouse retinal vasculature (FIG. 4(f)). Degeneration of the vasculature in rd/rd mouse eyes in observed asearly as 2-3 weeks post-natally. Injection of Lin⁻ HSC as late as P15also resulted in partial stabilization of the degenerating vasculaturein the rd/rd mice for at least one month (FIG. 4 (g and h)).

A Lin⁻ HSC composition injected into younger (e.g., P2) rd/rd mice alsoincorporated into the developing superficial vasculature. By P11, thesecells were observed to migrate to the level of the deep vascular plexusand form a pattern identical to that observed in the wild type outerretinal vascular layer (FIG. 5 (a)). In order to more clearly describethe manner in which cells from injected Lin⁻ HSC compositionsincorporate into, and stabilize, degenerating retinal vasculature in therd/rd mice, a Lin⁻ HSC composition derived from Balb/c mice was injectedinto Tie-2-GFP FVB mouse eyes. The FVB mice have the rd/rd genotype andbecause they express the fusion protein Tie-2-GFP, all endogenous bloodvessels are fluorescent.

When non-labeled cells from a Lin⁻ HSC composition are injected intoneonatal Tie-2-GFP FVB eyes and are subsequently incorporated into thedeveloping vasculature, there should be non-labeled gaps in theendogenous, Tie-2-GFP labeled vessels that correspond to theincorporated, non-labeled Lin⁻ HSC that was injected. Subsequentstaining with another vascular marker (e.g., CD-31) then delineates theentire vessel, permitting determination as to whether non-endogenousendothelial cells are part of the vasculature. Two months afterinjection, CD31-positive, Tie-2-GFP negative, vessels were observed inthe retinas of eyes injected with the Lin⁻ HSC composition (FIG. 5 (b)).Interestingly, the majority of rescued vessels contained Tie-2-GFPpositive cells (FIG. 5 (c)). The distribution of pericytes, asdetermined by staining for smooth muscle actin, was not changed by Lin⁻HSC injection, regardless of whether there was vascular rescue (FIG. 5(d)). These data clearly demonstrate that intravitreally injected Lin⁻HSC cells migrate into the retina, participate in the formation ofnormal retinal blood vessels, and stabilize endogenous degeneratingvasculature in a genetically defective mouse.

Inhibition of Retinal Angiogenesis by Transfected Cells from Lin⁻ HSC.

The majority of retinal vascular diseases involve abnormal vascularproliferation rather than degeneration. Transgenic cells targeted toastrocytes can be used to deliver an anti-angiogenic protein and inhibitangiogenesis. Cells from Lin⁻ HSC compositions were transfected withT2-tryptophanyl-tRNA synthetase (T2-TrpRS). T2-TrpRS is a 43 kD fragmentof TrpRS that potently inhibits retinal angiogenesis (FIG. 6 (a)). OnP12, retinas of eyes injected with a control plasmid-transfected Lin⁻HSC composition (no T2-TrpRS gene) on P2 had normal primary (FIG. 6 (c))and secondary (FIG. 6 (d)) retinal vascular plexuses. When the T2-TrpRStransfected Lin⁻ HSC composition of the present invention was injectedinto P2 eyes and evaluated 10 days later, the primary network hadsignificant abnormalities (FIG. 6 (e)) and formation of the deep retinalvasculature was nearly completely inhibited (FIG. 6 (f)). The fewvessels observed in these eyes were markedly attenuated with large gapsbetween vessels. The extent of inhibition by T2-TrpRS-secreting Lin⁻HSCs is detailed in Table 1.

T2-TrpRS is produced and secreted by cells in the Lin⁻ HSC compositionin vitro and after injection of these transfected cells into thevitreous, a 30 kD fragment of T2-TrpRS in the retina (FIG. 6 (b)) wasobserved. This 30 kD fragment was specifically observed only in retinasinjected with transfected Lin⁻ HSC and this decrease in apparentmolecular weight compared to the recombinant or in vitro-synthesizedprotein may be due to processing or degradation of the T2-TrpRS in vivo.These data indicate that Lin⁻ HSC compositions can be used to deliverfunctionally active genes, such as genes expressing angiostaticmolecules, to the retinal vasculature by targeting to activatedastrocytes. While it is possible that the observed angiostatic effect isdue to cell-mediated activity this is very unlikely since eyes treatedwith identical, but non-T2-transfected Lin⁻ HSC compositions had normalretinal vasculature.

TABLE 1 Vascular Inhibition by T2-TrpRS-secreting Lin⁻ HSCs PrimaryPlexus Deep Plexus Inhibited Normal Complete Partial Normal T2-TrpRS 60% 40% 33.3%   60%  6.7% (15 eyes) (9 eyes) (6 eyes) (5 eyes) (9 eyes) (1eye) Control  0% 100%   0% 38.5% 61.5% (13 eyes) (0 eyes) (13 eyes) (0eyes) (5 eyes) (8 eyes)

Intravitreally injected Lin⁻ HSC populations localize to retinalastrocytes, incorporate into vessels, and can be useful in treating manyretinal diseases. While most cells from injected HSC compositions adhereto the astrocytic template, small numbers migrate deep into the retina,homing to regions where the deep vascular network will subsequentlydevelop. Even though no GFAP-positive astrocytes were observed in thisarea prior to 42 days postnatally, this does not rule out thepossibility that GFAP-negative glial cells are already present toprovide a signal for Lin⁻ HSC localization. Previous studies have shownthat many diseases are associated with reactive gliosis. In DR, inparticular, glial cells and their extracellular matrix are associatedwith pathological angiogenesis.

Since cells from injected Lin⁻ HSC compositions specifically attached toGFAP-expressing glial cells, regardless of the type of injury, Lin⁻ HSCcompositions of the present invention can be used to targetpre-angiogenic lesions in the retina. For example, in the ischemicretinopathies, such as diabetes, neovascularization is a response tohypoxia. By targeting Lin⁻ HSC compositions to sites of pathologicalneovascularization, developing neovasculature can be stabilizedpreventing abnormalities of neovasculature such as hemorrhage or edema(the causes of vision loss associated with DR) and can potentiallyalleviate the hypoxia that originally stimulated the neovascularization.Abnormal blood vessels can be restored to normal condition. Furthermore,angiostatic proteins, such as T2-TrpRS can be delivered to sites ofpathological angiogenesis by using transfected Lin⁻ HSC compositions andlaser-induced activation of astrocytes. Since laser photocoagulation iscommonly used in clinical ophthalmology, this approach has applicationfor many retinal diseases. While such cell-based approaches have beenexplored in cancer therapy, their use for eye diseases is moreadvantageous since intraocular injection makes it possible to deliverlarge numbers of cells directly to the site of disease.

Neurotrophic and Vasculotrophic Rescue by Lin⁻ HSC.

MACS was used to separate Lin⁻ HSC from bone marrow of enhanced greenfluorescent protein (eGFP), C3H (rd/rd), FVB (rd/rd) mice as describedabove. Lin⁻ HSC containing EPC from these mice were injectedintravitreally into P6 C3H or FVB mouse eyes. The retinas were collectedat various time points (1 month, 2 months, and 6 months) afterinjection. The vasculature was analyzed by scanning laser confocalmicroscope after staining with antibodies to CD31 and retinal histologyafter nuclear staining with DAPI. Microarray gene expression analysis ofmRNA from retinas at varying time points was also used to identify genespotentially involved in the effect.

Eyes of rd/rd mice had profound degeneration of both neurosensory retinaand retinal vasculature by P21. Eyes of rd/rd mice treated with Lin⁻ HSCon P6 maintained a normal retinal vasculature for as long as 6 months;both deep and intermediate layers were significantly improved whencompared to the controls at all time points (1M, 2M, and 6M) (see FIG.12). In addition, we observed that retinas treated with Lin⁻ HSC werealso thicker (1M; 1.2-fold, 2M; 1.3-fold, 6M; 1.4-fold) and had greaternumbers of cells in the outer nuclear layer (1M; 2.2-fold, 2M; 3.7-fold,6M; 5.7-fold) relative to eyes treated with Lin⁺ HSC as a control. Largescale genomic analysis of “rescued” (e.g., Lin⁻ HSC) compared to control(untreated or non-Lin⁻ treated) rd/rd retinas demonstrated a significantupregulation of genes encoding sHSPs (small heat shock proteins) andspecific growth factors that correlated with vascular and neural rescue,including genes encoding the proteins listed in FIG. 20, panels A and B.

The bone marrow derived Lin⁻ HSC populations significantly andreproducibly induced maintenance of a normal vasculature anddramatically increased photoreceptor and other neuronal cell layers inthe rd/rd mouse. This neurotrophic rescue effect correlated withsignificant upregulation of small heat shock proteins and growth factorsand provides insights into therapeutic approaches to currentlyuntreatable retinal degenerative disorders.

Rd1/rd1 Mouse Retinas Exhibit Profound Vascular and NeuronalDegeneration.

Normal postnatal retinal vascular and neuronal development in mice hasbeen well described and is analogous to changes observed in the thirdtrimester human fetus (Dorrell et al., 2002, Invest. Ophthalmol. Vis.Sci. 43:3500-3510). Mice homozygous for the rd1 gene share manycharacteristics of human retinal degeneration (Frasson et al., 1999,Nat. Med. 5:1183-1187) and exhibit rapid photoreceptor (PR) lossaccompanied by severe vascular atrophy as the result of a mutation inthe gene encoding PR cGMP phosphodiesterase (Bowes et al. 1990, Nature347:677-680). To examine the vasculature during retinal development andits subsequent degeneration, antibodies against collagen IV (CIV), anextracellular matrix (ECM) protein of mature vasculature, and CD31(PECAM-1), a marker for endothelial cells, were used (FIG. 15). Retinasof rd1/rd1 (C3H/HeJ) developed normally until approximately postnatalday (P) 8 when degeneration of the photoreceptor-containing outernuclear layer (ONL) began. The ONL rapidly degenerated and cells died byapoptosis such that only a single layer of nuclei remained by P20.Double staining of the whole-mounted retinas with antibodies to both CIVand CD31 revealed details of the vascular degeneration in rd1/rd1 micesimilar to that described by others (Blanks et al., 1986, J. Comp.Neurol. 254:543-553). The primary and deep retinal vascular layersappeared to develop normally though P12 after which there is a rapidloss of endothelial cells as evidenced by the absence of CD31 staining.CD31 positive endothelial cells were present in a normal distributionthrough P12 but rapidly disappeared after that. Interestingly, CIVpositive staining remained present throughout the time points examined,suggesting that the vessels and associated ECM formed normally, but onlythe matrix remained after P13 by which time no CD31 positive cells wereobserved. (FIG. 15, middle panels). The intermediate vascular plexusalso degenerates after P21, but the progression is slower than thatobserved in the deep plexus (FIG. 15, upper panel). Retinal vascular andneural cell layers of a normal mouse are shown for comparison to therd1/rd1 mouse (right panels, FIG. 15).

Neuroprotective Effect of Bone Marrow-Derived Lin⁻ HSCs in rd1/rd1 Mice.

Intravitreally injected Lin⁻ HSCs incorporate into endogenous retinalvasculature in all three vascular plexuses and prevent the vessels fromdegenerating. Interestingly, the injected cells are virtually neverobserved in the outer nuclear layer. These cells either incorporate intothe forming retinal vessels or are observed in close proximity to thesevessels. Murine Lin⁻ HSCs (from C3H/HeJ) were intravitreally injectedinto C3H/HeJ (rd1/rd1) mouse eyes at P6, just prior to the onset ofdegeneration. By P30, control cell (CD31⁻)-injected eyes exhibited thetypical rd1/rd1 phenotype, i.e., nearly complete degeneration of thedeep vascular plexus and ONL was observed in every retina examined. Eyesinjected with Lin⁻ HSCs maintained normal-appearing intermediate anddeep vascular plexuses. Surprisingly, significantly more cells wereobserved in the internuclear layer (INL) and ONL of Lin⁻ HSC-injectedeyes than in control cell-injected eyes (FIG. 16 (A)). This rescueeffect of Lin⁻ HSCs could be observed at 2 months (FIG. 16 (B)) and foras long as 6 months after injection (FIG. 16 (C)). Differences in thevasculature of the intermediate and deep plexuses of Lin⁻ HSC-injectedeyes, as well as the neuronal cell-containing INL and ONL, weresignificant at all time points measured when rescued and non-rescuedeyes were compared (FIGS. 16 (B and C)). This effect was quantified bymeasuring the total length of the vasculature (FIG. 16 (D)) and countingthe number of DAPI-positive cell nuclei observed in the ONL (FIG. 16(E)). Simple linear-regression analysis was applied to the data at alltime points.

A statistically significant correlation was observed between vascularrescue and neuronal (e.g., ONL thickness) rescue at P30 (p<0.024) andP60 (p<0.034) in the Lin⁻ HSC-injected eyes (FIG. 16 (F)). Thecorrelation remained high, although not statistically significant(p<0.14) at P180 when comparing Lin⁻ HSC-injected retinas to controlcell-injected retinas (FIG. 16 (F)). In contrast, control cell-injectedretinas showed no significant correlation between the preservation ofvasculature and ONL at any time point (FIG. 16 (F)). These datademonstrate that intravitreal injection of Lin⁻ HSCs results inconcomitant retinal vascular and neuronal rescue in retinas of rd1/rd1mice. Injected cells were not observed in the ONL or any place otherthan within, or in close proximity to, retinal blood vessels.

Functional Rescue of Lin⁻ HSC-injected rd/rd Retinas.

Electroretinograms (ERGs) were performed on mice 2 months afterinjection of control cells or murine Lin⁻ HSCs (FIG. 17).Immunohistochemical and microscopic analysis was done with each eyefollowing ERG recordings to confirm that vascular and neuronal rescuehad occurred. Representative ERG recordings from treated, rescued andcontrol, non-rescued eyes show that in the rescued eyes, the digitallysubtracted signal (treated minus untreated eyes) produced a clearlydetectable signal with an amplitude on the order of 8-10 microvolts(FIG. 17). Clearly, the signals from both eyes are severely abnormal.However, consistent and detectable ERGs were recordable from the Lin⁻HSC-treated eyes. In all cases the ERG from the control eye wasnon-detectable. While the amplitudes of the signals in rescued eyes wereconsiderably lower than normal, the signals were consistently observedwhenever there was histological rescue and were on the order ofmagnitude of those reported by other, gene based, rescue studies.Overall these results are demonstrate of some degree of functionalrescue in the eyes treated with the Lin⁻ HSCs.

Rescued rd/rd retinal cell types are predominantly cones.

Rescued and non-rescued retinas were analyzed immunohistochemically withantibodies specific for rod or cone opsin. The same eyes used for theERG recordings presented in FIG. 17 were analyzed for rod or cone opsin.In wild type mouse retinas, less than about 5% of photoreceptors presentare cones (Soucy et al. 1998, Neuron 21: 481-493) and theimmunohistochemical staining patterns observed with red/green cone opsinas shown in FIG. 25 (A) or rod rhodopsin as shown in FIG. 25 (B), wereconsistent with this percentage of cone cells. When wild type retinaswere stained with pre-immune IgG, no staining was observed anywhere inthe neurosensory retinas other than autofluorescence of the bloodvessels (FIG. 25 (C)). Two months after birth, retinas of non-injectedrd/rd mice had an essentially atrophic outer nuclear layer that does notexhibit any staining with antibodies to red green cone opsin (FIG. 25(D)) or rhodopsin (FIG. 25 (G)). Eyes injected with control, CD31− HSCalso did not stain positively for the presence of either cone (FIG. 25(E))) or rod (FIG. 25 (H)) opsin. In contrast, contralateral eyesinjected with Lin-HSC had about 3 to about 8 rows of nuclei in apreserved outer nuclear layer; most of these cells were positive forcone opsin (FIG. 25 (F)) with approximately 1-3% positive for rod opsin(FIG. 25 (I)). Remarkably, this is nearly the reverse of what isordinarily observed in the normal mouse retina, which is rod-dominated.These data demonstrate that the injection of Lin-HSC preserves cones forextended periods of time during which they would ordinarily degenerate.

Human bone marrow (hBM)-derived Lin⁻ HSCs also Rescue DegeneratingRetinas.

Lin⁻ HSCs isolated from human bone marrow behave similarly to murineLin⁻ HSCs. Bone marrow was collected from human donors and the Lin⁺ HSCswere depleted, producing a population of human Lin⁻ HSCs (hLin⁻ HSCs).These cells were labeled ex-vivo with fluorescent dye and injected intoC3SnSmn.CB17-Prkdc SCID mouse eyes. The injected hLin⁻ HSCs migrated to,and targeted, sites of retinal angiogenesis in a fashion identical tothat observed when murine Lin⁻ HSCs were injected (FIG. 18 (A)). Inaddition to the vascular targeting, the human Lin⁻ HSCs also provided arobust rescue effect on both the vascular and neuronal cell layers ofthe rd1/rd1 mice (FIG. 18 (B and C)). This observation confirms thepresence of cells in human bone marrow that target retinal vasculatureand can prevent retinal degeneration.

Lin⁻ HSCs have Vasculo- and Neurotrophic Effects in the rd10/rd10 Mouse.

While the rd1/rd1 mouse is the most widely used and best characterizedmodel for retinal degeneration (Chang et al. 2002, Vision Res.42:517-525), the degeneration is very rapid and in this regard differsfrom the usual, slower time course observed in the human disease. Inthis strain, photoreceptor cell degeneration begins around P8, a timewhen the retinal vasculature is still rapidly expanding (FIG. 15).Subsequent degeneration of the deep retinal vasculature occurs evenwhile the intermediate plexus is still forming and, thus, the retinas ofrd1/rd1 mice never completely develops, unlike that observed in mosthumans with this disease. An rd10 mouse model, which has a slower timecourse of degeneration and more closely resembles the human retinaldegenerative condition, was used to investigate Lin⁻ HSC-mediatedvascular rescue. In the rd10 mouse, photoreceptor cell degenerationbegins around P21 and vascular degeneration begins shortly thereafter.

Since normal neurosensory retinal development is largely complete byP21, the degeneration is observed to start after the retina hascompleted differentiation and in this way is more analogous to humanretinal degenerations than the rd1/rd1 mouse model. Lin⁻ HSCs or controlcells from rd10 mice were injected into P6 eyes and the retinas wereevaluated at varying time points. At P21 the retinas from both Lin⁻ HSCand control cell-injected eyes appeared normal with complete developmentof all vascular layers and normal development of the INL and ONL (FIG.18 (D and H)). At approximately P21 the retinal degeneration began andprogressed with age. By P30, the control cell-injected retinas exhibitedsevere vascular and neuronal degeneration (FIG. 18 (I)), while the Lin⁻HSC-injected retinas maintained nearly normal vascular layers andphotoreceptor cells (FIG. 18 (E)). The difference between the rescuedand non-rescued eyes was more pronounced at later time points (compareFIGS. 18 (F and G) to 18 (J and K)). In the control treated eyes, theprogression of vascular degeneration was very clearly observed byimmunohistochemical staining for CD31 and collagen IV (FIG. 18 (I-K)).The control-treated eyes were nearly completely negative for CD31,whereas collagen IV-positive vascular “tracks” remained evident,indicating that vascular regression, rather than incomplete vascularformation, had occurred. In contrast, Lin⁻ HSC-treated eyes had bothCD31 and collagen IV-positive vessels that appeared very similar tonormal, wild-type eyes (compare FIG. 18 (F and I)).

Gene Expression Analysis of rd/rd Mouse Retinas after Lin⁻ HSCTreatment.

Large scale genomics (microarray analysis) was used to analyze rescuedand non-rescued retinas to identify putative mediators of neurotrophicrescue. Gene expression in rd1/rd1 mouse retinas treated with Lin⁻ HSCswas compared to uninjected retinas as well as retinas injected withcontrol cells (CD31⁻). These comparisons each were performed intriplicate. To be considered present, genes were required to haveexpression levels at least 2-fold higher than background levels in allthree triplicates. Genes that were upregulated 3-fold in Lin⁻HSC-protected retinas compared to control cell-injected and non-injectedrd/rd mouse retinas are shown in FIG. 20, panels A and B. Coefficient ofvariance (COV) levels were calculated for the expressed genes bydividing the standard deviation by the mean expression level of eachcRNA replicate. In addition, the correlation between expression levelsand noise variance was calculated by correlating the mean and standarddeviation (SD). A correlation between gene expression level and standarddeviation for each gene was obtained, allowing background levels andreliable expression level thresholds to be determined. As a whole, thedata fell well within acceptable limits (Tu et al. 2002, Proc. Natl.Acad. Sci. USA 99: 14031-14036). The genes that are discussedindividually, below, exhibited expression levels above these criticalexpression levels. Paired “t-test” values for the discussed genes werealso determined. In each case, p-values are reasonable (near or below0.05), which demonstrates that there are similarities between replicatesand probable significant differences between the different test groups.Many of the significantly upregulated genes, including MAD and YingYang-1 (YY-1) (Austen et al. 1997, Curr. Top. Microbiol. Immunol. 224:123-130), encode proteins with functions involving the protection ofcells from apoptosis. A number of crystallin genes, which have sequencehomology and similar functions to known heat-shock proteins involvingprotection of cells from stress, were also upregulated by Lin− HSCtreatment. Expression of α-crystallin was localized to the ONL byimmunohistochemical analysis (FIG. 19). FIG. 19 shows that crystallin αAis upregulated in rescued outer nuclear layer cells after treatment withLin⁻ HSCs but not in contralateral eyes treated with control cells. Theleft panel shows IgG staining (control) in rescued retina. The middlepanel shows crystallin αA in a rescued retina. The right panel showscrystallin αA in non-rescued retina.

Messenger RNA from rd1/rd1 mouse retinas rescued with human Lin⁻ HSCswere hybridized to human specific Affymetrix U133A microarray chips.After stringent analysis, a number of genes were found whose mRNAexpression was human specific, above background, and significantlyhigher in the human Lin⁻ HSC rescued retinas compared to the murine Lin⁻HSC rescued retinas and the human control cell-injected non-rescuedretinas (FIG. 20, panel C). CD6, a cell adhesion molecule expressed atthe surface of primitive and newly differentiated CD34+ hematopoieticstem cells, and interferon alpha 13, another gene expressed byhematopoietic stem cells, were both found by the microarraybioinformatics technique, validating the evaluation protocol. Inaddition, several growth factors and neurotrophic factors were expressedabove background by human Lin⁻ HSC rescued mouse retina samples (FIG.20, panel D).

Markers for lineage-committed hematopoietic cells were used tonegatively select a population of bone marrow-derived Lin⁻ HSCcontaining EPC. While the sub-population of bone marrow-derived Lin⁻ HSCthat can serve as EPC is not characterized by commonly used cell surfacemarkers, the behavior of these cells in developing or injured retinalvasculature is entirely different than that observed for Lin⁺ or adultendothelial cell populations. These cells selectively target to sites ofretinal angiogenesis and participate in the formation of patent bloodvessels.

Inherited retinal degenerative diseases are often accompanied by loss ofretinal vasculature. Effective treatment of such diseases requiresrestoration of function as well as maintenance of complex tissuearchitecture. While several recent studies have explored the use ofcell-based delivery of trophic factors or stem cells themselves, somecombination of both may be necessary. For example, use of growth factortherapy to treat retinal degenerative disease resulted in unregulatedovergrowth of blood vessels resulting in severe disruption of the normalretinal tissue architecture. The use of neural or retinal stem cells totreat retinal degenerative disease may reconstitute neuronal function,but a functional vasculature will also be necessary to maintain retinalfunctional integrity. Incorporation of cells from a Lin⁻ HSC populationinto the retinal vessels of rd/rd mice stabilized the degenerativevasculature without disrupting retinal structure. This rescue effect wasalso observed when the cells were injected into P15 rd/rd mice. Sincevascular degeneration begins on P16 in rd/rd mice, this observationexpands the therapeutic window for effective Lin⁻ HSC treatment. Retinalneurons and photoreceptors are preserved and visual function ismaintained in eyes injected with the Lin⁻ HSC cells.

Adult bone marrow-derived Lin⁻ HSCs exert profound vasculo- andneurotrophic effects when injected intravitreally into mice with retinaldegenerative disease. This rescue effect persists for up to 6 monthsafter treatment and is most efficacious when the Lin⁻ HSCs are injectedprior to complete retinal degeneration (up to 16 days after birth inmice that ordinarily exhibit complete retinal degeneration by 30 dayspostnatally). This rescue is observed in two mouse models of retinaldegeneration and, remarkably, can be accomplished with adult human bonemarrow-derived HSCs when the recipient is an immunodeficient rodent withretinal degeneration (e.g., the SCID mouse) or when the donor is a mousewith retinal degeneration. While several recent reports have described apartial phenotypic rescue in mice or dogs with retinal degenerationafter viral based gene rescue with the wild type gene (Ali, et al. 2000,Nat Genet. 25:306-310; Takahashi et al. 1999, J. Virol. 73:7812-7816;Acland et al. 2001, Nat. Genet. 28:92-95), the present invention is thefirst generic cell-based therapeutic effect achieved by vascular rescue.Thus, the potential utility of such an approach in treating a group ofdiseases (e.g., retinitis pigmentosa) with over 100 known associatedmutations is more practical than creating individual gene therapies totreat each known mutation.

The precise molecular basis of the neurotrophic rescue effect remainsunknown, but is observed only when there is concomitant vascularstabilization/rescue. The presence of injected stem cells, per se, isnot sufficient to generate a neurotrophic rescue and the clear absenceof stem cell-derived neurons in the outer nuclear layer rules out thepossibility that the injected cells are transforming intophotoreceptors. Data obtained by microarray gene expression analysisdemonstrated a significant up-regulation of genes known to haveanti-apoptotic effects. Since most neuronal death observed in retinaldegenerations is by apoptosis, such protection may be of greattherapeutic benefit in prolonging the life of photoreceptors and otherneurons critical to visual function in these diseases. C-myc is atranscription factor that participates in apoptosis by upregulation ofvarious downstream apoptosis-inducing factors. C-myc expression wasincreased 4.5 fold in rd/rd mice over wild-type indicating potentialinvolvement in the photoreceptor degeneration observed in the rd1/rd1mouse. Mad1 and YY-1, two genes dramatically upregulated in Lin⁻HSC-protected retinas (FIG. 20, panel A), are known to suppress theactivity of c-myc, thus inhibiting c-myc induced apoptosis.Overexpression of Mad1 has also been shown to suppress Fas-inducedactivation of caspase-8, another critical component of the apoptoticpathway. Upregulation of these two molecules may play a role inprotection of the retina from vascular and neural degeneration bypreventing the initiation of apoptosis that normally leads todegeneration in rd/rd mice.

Another set of genes that were greatly upregulated in Lin⁻ HSC protectedretinas includes members of the crystallin family (FIG. 20, panel B).Similar to heat-shock and other stress-induced proteins, crystallins maybe activated by retinal stress and provide a protective effect againstapoptosis. Abnormally low expression of crystallin αA is correlated withphotoreceptor loss in a rat model of retinal dystrophy and a recentproteomic analysis of the retina in the rd/rd mouse demonstratedinduction of crystallin upregulation in response to retinaldegeneration. Based on our microarray data of EPC-rescued rd/rd mouseretinas, upregulation of crystallins appear to play a key role in EPCmediated retinal neuroprotection.

Genes such as c-myc, Mad1, Yx-1 and the crystallins are likely to bedownstream mediators of neuronal rescue. Neurotrophic agents canregulate anti-apoptotic gene expression, although our microarrayanalysis of retinas rescued with mouse stem cells did not demonstrateinduction of increased levels of known neurotrophic factors. Analysis ofhuman bone marrow-derived stem cell-mediated rescue with human specificchips did, on the other hand, demonstrate low, but significant increasesin the expression of multiple growth factor genes.

The upregulated genes include several members of the fibroblast growthfactor family and otoferlin. Mutations in the otoferlin gene areassociated with genetic disorders leading to deafness due to auditoryneuropathy. It is possible that otoferlin production by injected Lin⁻HSCs contributes to the prevention of retinal neuropathy as well.Historically, it has long been assumed that vascular changes observed inpatients and animals with retinal degeneration were secondary todecreased metabolic demand as the photoreceptors die. The present dataindicate that, at least for mice with inherited retinal degeneration,preserving normal vasculature can help maintain components of the outernuclear layer as well. Recent reports in the literature would supportthe concept that tissue-specific vasculature has trophic effects that gobeyond that expected from simply providing vascular “nourishment.” Forexample, liver endothelial cells can be induced to produce, after VEGFR1activation, growth factors critical to hepatocyte regeneration andmaintenance in the face of hepatic injury (LeCouter et al. 2003, Science299:890-893).

Similar indicative interactions between vascular endothelial cells andadjacent hepatic parenchymal cells are reportedly involved in liverorganogenesis, well before the formation of functional blood vessels.Endogenous retinal vasculature in individuals with retinal degenerationmay not facilitate so dramatic a rescue, but if this vasculature isbuttressed with endothelial progenitors derived from bone marrowhematopoietic stem cell populations, they may make the vasculature moreresistant to degeneration and at the same time facilitate retinalneuronal, as well as vascular, survival. In humans with retinaldegeneration, delaying the onset of complete retinal degeneration mayprovide years of additional sight. The animals treated with Lin⁻ HSCshad significant preservation of an ERG, which may be sufficient tosupport vision.

Clinically, it is widely appreciated that there may be substantial lossof photoreceptors and other neurons while still preserving functionalvision. At some point, the critical threshold is crossed and vision islost. Since nearly all of the human inherited retinal degenerations areof early, but slow, onset, an individual with retinal degeneration canbe identified and treated intravitreally with a graft of autologous bonemarrow stem cells of the invention to delay retinal degeneration andconcomitant loss of vision. To enhance targeting and incorporation ofthe stem cells of the invention, the presence of activated astrocytes isdesirable (Otani et al. 2002, Nat. Med. 8: 1004-1010); this can beaccomplished by early treatment when there is an associated gliosis, orby using a laser to stimulate local proliferation of activatedastrocytes. Optionally, ex vivo transfection of the stem cells with oneor more neurotrophic substances prior to intraocular injection can beused to enhance the rescue effect. This approach can be applied to thetreatment of other visual neuronal degenerative disorders, such asglaucoma, in which there is retinal ganglion cell degeneration.

The Lin⁻ HSC populations from adult bone marrow contain a population ofEPC that can promote angiogenesis by targeting reactive astrocytes andincorporate into an established template without disrupting retinalstructure. The Lin⁻ HSC also provide a long-term neurotrophic rescueeffect in eyes suffering from retinal degeneration. In addition,genetically modified, autologous Lin⁻ HSC compositions containing EPCcan be transplanted into ischemic or abnormally vascularized eyes andcan stably incorporate into new vessels and neuronal layers andcontinuously deliver therapeutic molecules locally for prolonged periodsof time. Such local delivery of genes that express pharmacologicalagents in physiologically meaningful doses represents a new paradigm fortreating currently untreatable ocular diseases.

Photoreceptors in the normal mouse retina, for example, arepredominantly rods, but the outer nuclear layer observed after rescuewith Lin-HSCs of the invention contained predominantly cones. Mostinherited human retinal degenerations occur as a result of primaryrod-specific defects, and loss of the cones is believed to be secondaryto rod dysfunction, which is likely related to the loss of some trophicfactor expressed by rods.

EXAMPLES Example 1 Cell Isolation and Enrichment; Preparation of MurineLin⁻ HSC Populations A and B

General Procedure. All in vivo evaluations were performed in accordancewith the NIH Guide for the Care and Use of Laboratory Animals, and allevaluation procedures were approved by The Scripps Research Institute(TSRI, La Jolla, Calif.) Animal Care and Use Committee. Bone marrowcells were extracted from B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB/NJ(rd/rd mice) or Balb/cBYJ adult mice (The Jackson Laboratory, ME).

Monocytes were then separated by density gradient separation usingHISTOPAQUE™ polysucrose gradient (Sigma, St. Louis, Mo.) and labeledwith biotin conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD11,TER-119, Pharmingen, San Diego, Calif.) for Lin⁻ selection in mice.Lineage positive (Lin⁺) cells were separated and removed from Lin⁻ HSCusing a magnetic separation device (AUTOMACS™ sorter, Miltenyi Biotech,Auburn, Calif.). The resulting Lin⁻ HSC population, containingendothelial progenitor cells was further characterized using a FACS™Calibur flow cytometer (Becton Dickinson, Franklin Lakes, N.J.) usingthe following antibodies: PE-conjugated-Sca-1, c-kit, KDR, and CD31(Pharmingen, San Diego, Calif.). Tie-2-GFP bone marrow cells were usedfor the characterization of Tie-2.

To harvest adult mouse endothelial cells, mesenteric tissue wassurgically removed from ACTbEGFP mouse and placed in collagenase(Worthington, Lakewood, N.J.) to digest the tissue, followed byfiltration using a 45 μm filter. Flow-through was collected andincubated with Endothelial Growth Media (Clonetics, San Diego, Calif.).Endothelial characteristics were confirmed by observing morphologicalcobblestone appearance, staining with CD31 mAb (Pharmingen) andexamining cultures for the formation of tube-like structures inMATRIGEL™ matrix (Beckton Dickinson, Franklin Lakes, N.J.).

Murine Lin⁻ HSC Population A. Bone marrow cells were extracted fromACTbEGFP mice by the General Procedure described above. The Lin⁻ HSCcells were characterized by FACS flow cytometry for CD31, c-kit, Sca-1,Flk-1, and Tie-2 cell surface antigen markers. The results are shown inFIG. 1 (c). About 81% of the Lin⁻ HSC exhibited the CD31 marker, about70.5% of the Lin⁻ HSC exhibited the c-kit marker, about 4% of the Lin⁻HSC exhibited the Sca-1 marker, about 2.2% of the Lin⁻ HSC exhibited theFlk-1 marker and about 0.91% of the Lin⁻ HSC cell exhibited the Tie-2marker. In contrast, the Lin⁺ HSC that were isolated from these bonemarrow cells had a significantly different cell marker profile (i.e.,CD31: 37.4%; c-kit: 20%; Sca-1: 2.8%; Flk-: 0.05%).

Murine Lin⁻ HSC Population B. Bone marrow cells were extracted fromBalb/C, ACTbEGFP, and C3H mice by the General Procedure described above.The Lin⁻ HSC cells were analyzed for the presence of cell surfacemarkers (Sca-1, Flk-1/KDR, c-kit (CD117), CD34, CD31 and variousintegrins: α1, α2, α3, α4, α5, α6, α_(L), α_(M) α_(V), α_(X), α_(IIb),β₁, β₂, β₃, β₄, β₅ and β₇). The results are shown in Table 2.

TABLE 2 Characterization of Lin⁻ HSC Population B. Cell Marker Lin⁻ HSCα1 0.10 α2 17.57 α3 0.22 α4 89.39 α5 82.47 α6 77.70 αL 62.69 αM 35.84 αX3.98 αV 33.64 αIIb 0.25 β1 86.26 β2 49.07 β3 45.70 β4 0.68 β5 9.44 β711.25 CD31 51.76 CD34 55.83 Flk-1/KDR 2.95 c-kit (CD117) 74.42 Sca-17.54

Example 2 Intravitreal Administration of Cells in a Murine Model

An eyelid fissure was created in a mouse eyelid with a fine blade toexpose the P2 to P6 eyeball. Lineage negative HSC Population A of thepresent invention (approximately 10⁵ cells in about 0.5 μl to about 1 μlof cell culture medium) was then injected intravitreally using a33-gauge (Hamilton, Reno, Nev.) needled-syringe.

Example 3 EPC Transfection

Murine Lin⁻ HSC (Population A) were transfected with DNA encoding the T2fragment of TrpRS also enclosing a His₆ tag (SEQ ID NO: 1, FIG. 7) usingFuGENE™ 6 Transfection Reagent (Roche, Indianapolis, Ind.) according tomanufacturer's protocol. Lin⁻ HSC cells (about 10⁶ cell per ml) weresuspended in OPTI-MEM® medium (Invitrogen, Carlsbad, Calif.) containingstem cell factor (PeproTech, Rocky Hill, N.J.). DNA (about 1 μg) andFuGENE reagent (about 3 μl) mixture was then added, and the mixtureswere incubated at about 37° C. for about 18 hours. After incubation,cells were washed and collected. The transfection rate of this systemwas approximately 17% as confirmed by FACS analysis. T2-TrpRS productionwas confirmed by western blotting. The amino acid sequence ofHis₆-tagged T2-TrpRS is shown as SEQ ID NO: 2, FIG. 8.

Example 4 Immunohistochemistry and Confocal Analysis

Mouse retinas were harvested at various time points and were preparedfor either whole mounting or frozen sectioning. For whole mounts,retinas were fixed with 4% paraformaldehyde, and blocked in 50% fetalbovine serum (FBS) and 20% normal goat serum for one hour at ambientroom temperature. Retinas were processed for primary antibodies anddetected with secondary antibodies. The primaries used were:anti-Collagen IV (Chemicon, Temecula, Calif., anti-β-gal (Promega,Madison, Wis.), anti-GFAP (Dako Cytomation, Carpenteria, Calif.),anti-α-smooth muscle actin (α-SMA, Dako Cytomation). Secondaryantibodies used were conjugated either to Alexa 488 or 594 fluorescentmarkers (Molecular Probes, Eugene, Oreg.). Images were taken using anMRC 1024 Confocal microscope (Bio-Rad, Hercules, Calif.).Three-dimensional images were created using LASERSHARP® software(Bio-Rad) to examine the three different layers of vascular developmentin the whole mount retina. The difference in GFP pixel intensity betweenenhanced GFP (eGFP) mice and GFAP/wtGFP mice, distinguished by confocalmicroscopy, was utilized to create the 3 dimensional images.

Example 5 In Vivo Retinal Angiogenesis Quantification Assay in Mice

For T2-TrpRS analysis, the primary and deep plexus were reconstructedfrom the three dimensional images of mouse retinas. The primary plexuswas divided into two categories: normal development, or halted vascularprogression. The categories of inhibition of deep vascular developmentwere construed based upon the percentage of vascular inhibitionincluding the following criteria: complete inhibition of deep plexusformation was labeled “Complete”, normal vascular development (includingless than 25% inhibition) was labeled “Normal” and the remainder labeled“Partial.” For the rd/rd mouse rescue data, four separate areas of thedeeper plexus in each whole mounted retina was captured using a 10×lens. The total length of vasculature was calculated for each image,summarized and compared between the groups. To acquire accurateinformation, Lin⁻ HSC were injected into one eye and Lin⁺ HSC intoanother eye of the same mouse. Non-injected control retinas were takenfrom the same litter.

Example 6 Adult Retinal Injury Murine Models

Laser and scar models were created using either a diode laser (150 mW, 1second, 50 mm) or mechanically by puncturing the mouse retina with a 27gauge needle. Five days after injury, cells were injected using theintravitreal method. Eyes were harvested from the mice five days later.

Example 7 Neurotrophic Rescue of Retinal Regeneration

Adult murine bone marrow derived lineage negative hematopoietic stemcells (Lin⁻ HSC) have a vasculotrophic and neurotrophic rescue effect ina mouse model of retinal degeneration. Right eyes of 10-day old micewere injected intravitreally with about 0.5 microliters containing about10⁵ Lin⁻ HSC of the present invention and evaluated 2 months later forthe presence of retinal vasculature and neuronal layer nuclear count.The left eyes of the same mice were injected with about the same numberof Lin⁺ HSC as a control, and were similarly evaluated. As shown in FIG.9, in the Lin⁻ HSC treated eyes, the retinal vasculature appeared nearlynormal, the inner nuclear layer was nearly normal and the outer nuclearlayer (ONL) had about 3 to about 4 layers of nuclei. In contrast, thecontralateral Lin⁺ HSC treated eye had a markedly atrophic middleretinal vascular layer, a completely atrophic outer retinal vascularlayer; the inner nuclear layer was markedly atrophic and the outernuclear layer was completely gone. This was dramatically illustrated inMouse 3 and Mouse 5. In Mouse 1, there was no rescue effect and this wastrue for approximately 15% of the injected mice.

When visual function was assessed with electroretinograms (ERG), therestoration of a positive ERG was observed when both the vascular andneuronal rescue was observed (Mice 3 and 5). Positive ERG was notobserved when there was no vascular or neuronal rescue (Mouse 1). Thiscorrelation between vascular and neurotrophic rescue of the rd/rd mouseeyes by the Lin⁻ HSC of the present invention is illustrated by aregression analysis plot shown in FIG. 10. A correlation betweenneuronal (y-axis) and vascular (x-axis) recovery was observed for theintermediate vasculature type (r=0.45) and for the deep vasculature(r=0.67).

FIG. 11 shows the absence of any statistically significant correlationbetween vascular and neuronal rescue by Lin⁺ HSC. The vascular rescuewas quantified and the data are presented in FIG. 12. Data for mice at 1month (1M), 2 months (2M), and 6 months (6M), post-injection shown inFIG. 12, demonstrate that vascular length was significantly increased ineyes treated with the Lin⁻ HSC of the present invention (dark bars)relative to the vascular length in untreated eyes from the same mouse(light bars), particularly at 1 month and 2 months, post-injection. Theneurotrophic rescue effect was quantified by counting nuclei in theinner and outer nuclear layers about two months after injection of Lin⁻HSC or Lin⁺ HSC. The results are presented in FIGS. 13 and 14.

Example 8 Human Lin⁻ HSC Population

Bone marrow cells were extracted from healthy adult human volunteers bythe General Procedure described above. Monocytes were then separated bydensity gradient separation using HISTOPAQUE® polysucrose gradient(Sigma, St. Louis, Mo.). To isolate the Lin⁻ HSC population from humanbone marrow mononuclear cells the following biotin conjugated lineagepanel antibodies were used with the magnetic separation system(AUTOMACS™ sorter, Miltenyi Biotech, Auburn, Calif.): CD2, CD3, CD4,CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86,CD235a (Pharmingen).

The human Lin⁻ HSC population was further separated into twosub-populations based on CD133 expression. The cells were labeled withbiotin-conjugated CD133 antibodies ans separated into CD133 positive andCD133 negative sub-populations.

Example 9 Intravitreal Administration of Human and Murine Cells inMurine Models for Retinal Degeneration

C3H/HeJ, C3SnSmn.CB17-Prkdc SCID, and rd10 mouse strains were used asretinal degeneration models. C3H/HeJ and C3SnSmn.CB17-Prkdc SCID mice(The Jackson Laboratory, Maine) were homozygous for the retinaldegeneration 1 (rd1) mutation, a mutation that causes early onset severeretinal degeneration. The mutation is located in exon 7 of the Pde6bgene encoding the rod photoreceptor cGMP phosphodiesterase β subunit.The mutation in this gene has been found in human patients withautosomal recessive retinitis pigmentosa (RP). C3SnSmn.CB17-Prkdc SCIDmice are also homozygous for the severe combined immune deficiencyspontaneous mutation (Prkdc SCID) and were used for human cell transferexperiments. Retinal degeneration in rd10 mice is caused by a mutationin exon 13 of Pde6b gene. This is also a clinically relevant RP modelwith later onset and milder retinal degeneration than rd1/rd1). Allevaluations were performed in accordance with the NIH Guide for the Careand Use of Laboratory Animals, and all procedures were approved by TheScripps Research Institute Animal Care and Use Committee.

An eyelid fissure was created in a mouse eyelid with a fine blade toexpose the P2 to P6 eyeball. Lineage negative HSC cells for murinepopulation A or human population C (approximately 10⁵ cells in about 0.5μl to about 1 μl of cell culture medium) were then injected in the mouseeye intravitreally using a 33-gauge (Hamilton, Reno, Nev.)needled-syringe. To visualize the injected human cells, cells werelabeled with dye (Cell tracker green CMFDA, Molecular Probes) beforeinjection.

Retinas were harvested at various time points and fixed with 4%paraformaldehyde (PFA) and methanol followed by blocking in 50% FBS/20%NGS for one hour at room temperature. To stain retinal vasculature,retinas were incubated with anti-CD31 (Pharmingen) and anti-collagen IV(Chemicon) antibodies followed by Alexa 488 or 594 conjugated secondaryantibodies (Molecular Probes, Eugene, Oreg.). The retinas were laid flatwith four radial relaxing incisions to obtain a whole mount preparation.Images of vasculature in intermediate or deep retinal vascular plexuses(see Dorrell et al. 2002 Invest Ophthalmol. Vis. Sci. 43:3500-3510) wereobtained using a Radiance MP2100 confocal microscope and LASERSHARP®software (Biorad, Hercules, Calif.). For quantification of vasculature,four independent fields (900 μm×900 μm) were chosen randomly from themid portion of the intermediate or deep vascular layer and the totallength of vasculature was measured using LASERPIX® analyzing software(Biorad). The total lengths of these four fields in the same plexus wereused for further analysis.

The flat-mounted retinas were re-embedded for cryostat sections. Retinaswere placed in 4% PFA overnight followed by incubation with 20% sucrose.The retinas were embedded in optimal cutting temperature compound (OCT:Tissue-Tek; Sakura FineTech, Torrance, Calif.). Cryostat sections (10μm) were re-hydrated in PBS containing the nuclear dye DAPI(Sigma-Aldrich, St. Louis, Mo.). DAPI-labeled nuclear images of threedifferent areas (280 μm width, unbiased sampling) in a single sectionthat contained optic nerve head and the entire peripheral retina weretaken by confocal microscope. The numbers of the nuclei located in ONLof the three independent fields in one section were counted and summedup for analysis. Simple linear-regression analysis was performed toexamine the relationship between the length of vasculature in the deepplexus and the number of cell nuclei in the ONL.

Following overnight dark-adaptation, mice were anesthetized byintraperitoneal injection of 15 μg/gm ketamine and 7 μg/gm xylazine.Electroretinograms (ERGs) were recorded from the corneal surface of eacheye after pupil dilation (1% atropine sulfate) using a gold loop cornealelectrode together with a mouth reference and tail ground electrode.Stimuli were produced with a Grass Photic Stimulator (PS33 Plus, GrassInstruments, Quincy, Mass.) affixed to the outside of a highlyreflective Ganzfeld dome. Rod responses were recorded toshort-wavelength (Wratten 47A; λ_(max)=470 nm) flashes of light over arange of intensities up to the maximum allowable by the photicstimulator (0.668 cd-s/m²). Response signals were amplified (CP511 ACamplifier, Grass Instruments), digitized (PCI-1200, NationalInstruments, Austin, Tex.) and computer-analyzed. Each mouse served asits own internal control with ERGs recorded from both the treated anduntreated eyes. Up to 100 sweeps were averaged for the weakest signals.The averaged responses from the untreated eye were digitally subtractedfrom the responses from the treated eye and this difference in signalwas used to index functional rescue.

Microarray analysis was used for evaluation of Lin⁻ HSC-targeted retinalgene expression. P6 rd/rd mice were injected with either Lin⁻ or CD31⁻HSCs. The retinas of these mice were dissected 40 days post-injection inRNase free medium (rescue of the retinal vasculature and thephotoreceptor layer is obvious at this time point after injection). Onequadrant from each retina was analyzed by whole mount to ensure thatnormal HSC targeting as well as vasculature and neural protection hadbeen achieved. RNA from retinas with successful injections was purifiedusing a TRIzol (Life Technologies, Rockville, Md.), phenol/chloroformRNA isolation protocol. RNA was hybridized to Affymetrix Mu74Av2 chipsand gene expression was analyzed using GENESPRING® software(SiliconGenetics, Redwood City, Calif.). Purified human or mouse HSCswere injected intravitreally into P6 mice. At P45 the retinas weredissected and pooled into fractions of 1) human HSC-injected, rescuedmouse retinas, 2) human HSC-injected, non-rescued mouse retinas, and 3)mouse HSC-injected, rescued mouse retinas for purification of RNA andhybridization to human-specific U133A Affymetrix chips. GENESPRING®software was used to identify genes that were expressed above backgroundand with higher expression in the human HSC-rescued retinas. Theprobe-pair expression profiles for each of these genes were thenindividually analyzed and compared to a model of normal human U133Amicroarray experiments using dChip to determine human species specifichybridization and to eliminate false positives due to cross-specieshybridization.

FIG. 21 illustrates flow cytometry data comparing the expression of CD31and integrin alpha 6 surface antigens on CD133 positive (CD133⁺) andCD133 negative (CD133⁻) human Lin⁻ HSC populations of the presentinvention. The left panels show flow cytometry scatter plots. The centerand right panels are histograms showing the level of specific antibodyexpression on the cell population. The Y axis represents the number ofevents and the X axis shows the intensity of the signal. The outlinedhistograms are isotype IgG control antibodies showing the level ofnon-specific background staining. The filled histograms show the levelof specific antibody expression on the cell population. A filledhistogram shifted to the right of the outlined (control) histogramrepresents an increased fluorescent signal and expression of theantibody above background level. Comparing the position of the peaks ofthe filled histograms between the two cell populations represents thedifference in protein expression on the cells. For example, CD31 isexpressed above background on both CD133⁺ and CD133⁻ cells of theinvention; however, there are more cells expressing lower levels of CD31in the CD133⁺ cell population than there are in the CD133⁻ population.From this data it is evident that CD31 expression varies between the twopopulations and that the alpha 6 integrin expression is largely limitedto cells in the Lin⁻ population, and thus may serve as a marker of cellswith vasculo- and neurotrophic rescue function.

When the CD133 positive and CD133 negative Lin⁻ HSC sub-population wasintravitreally injected into the eyes of neonatal SCID mice, thegreatest extent of incorporation into the developing vasculature wasobserved for the CD133 negative sub-population, which expresses bothCD31 and integrin α6 surface antigens (see FIG. 21, bottom). The CD133positive sub-population, which does not express CD31 or integrin α6(FIG. 21, top) appears to target sites of peripheral ischemia-drivenneovascularization, but not when injected into eyes undergoingangiogenesis.

Rescued and non-rescued retinas were analyzed immunohistochemically withantibodies specific for rod or cone opsin. The same eyes used for theERG recordings presented in FIG. 17 were analyzed for rod or cone opsin.In wild type mouse retinas, less than 5% of photoreceptors present arecones (Soucy et al. 1998, Neuron 21: 481-493) and theimmunohistochemical staining patterns observed with red/green cone opsinas shown in FIG. 25 (A) or rod rhodopsin as shown in FIG. 25 (B), wereconsistent with this percentage of cone cells. Antibodies specific forrod rhodopsin (rho4D2) were provided by Dr. Robert Molday of theUniversity of British Columbia and used as described previously (Hickset al. 1986, Exp. Eye Res. 42: 55-71). Rabbit antibodies specific forcone red/green opsin were purchased from Chemicon (AB5405) and usedaccording to the manufacturer's instructions.

Example 10 Intravitreal Administration of Murine Cells in Murine Modelsfor Oxygen Induced Retinal Degeneration

New born wild-type C57B16 mice were exposed to hyperoxia (75% oxygen)between postnatal days P7 to P12 in an oxygen-induced retinaldegeneration (OIR) model. FIG. 22 illustrates normal postnatal vasculardevelopment in C57B16 mice from P0 to P30. At P0 only buddingsuperficial vessels can be observed around the optic disc. Over the nextfew days, the primary superficial network extends toward the periphery,reaching the far periphery by day P10. Between P7 and P12, the secondary(deep) plexus develops. By P17, an extensive superficial and deepnetwork of vessels is present (FIG. 22, insets). In the ensuing days,remodeling occurs along with development of the tertiary (intermediate)layer of vessels until the adult structure is reached approximately atP21.

In contrast, in the OIR model described herein, following exposure to75% oxygen at P7-P12, the normal sequence of events is severelydisrupted (FIG. 23). Adult murine Lin⁻ HSC populations of the inventionwere intravitreally injected at P3 in an eye of a mouse that wassubsequently subjected to OIR, the other eye was injected with PBS orCD31 negative cells as a control. FIG. 24 illustrates that the Lin⁻ HSCpopulations can reverse the degenerative effects of high oxygen levelsin the developing mouse retina. Fully developed superficial and deepretinal vasculature was observed at P17 in the treated eyes, whereas thecontrol eyes showed large avascular areas with virtually no deep vessels(FIG. 24). Approximately 100 eyes of mice in the OIR model wereobserved. Normal vascularization was observed in 58% of the eyes treatedwith the Lin⁻ HSC populations, compared to 12% of the control eyestreated with CD31⁻ cells and 3% of the control eyes treated with PBS.

Example 11 Isolation of Myeloid-Like Bone Marrow Cells from Murine BoneMarrow by CD44 Selection

Bone marrow cells were extracted from adult mice (The JacksonLaboratory, ME). The whole bone marrow was treated with a murine CD44antibody and flow cytometry was used to isolate CD44 expressing cellsfrom the bone marrow. The cells were separated from the antibody andstored in a buffer solution for future use. A population of cells thatdo not significantly express CD44 was also isolated (CD44^(lo)BM).

Example 12 Isolation of Myeloid-Like Bone Marrow Cells From Murine BoneMarrow by CD44 Selection

Bone marrow cells were also positively selected using an antibody toCD11b in place of CD44, as described in Example 11. A myeloid-like bonemarrow cell population that was CD44^(hi) and CD11b+ was isolated, whichhad similar activity characteristics to the CD44^(hi) populationisolated in Example 11 using CD44. A CD44^(lo) CD11b⁻ population wasalso isolated, which was found to be inactive.

Example 13 Characterization of the MLBM Cell Populations

Although the role of CD44 in this context is not clear, it is possiblethat this receptor mediates cell survival, cell migration and/or celldifferentiation in the hyaluronic acid-rich vitreous following injectionof cells into the eye. Distinct populations of CD44^(hi) (i.e., MLBM)and CD44^(lo) cells were present in unfractionated mouse bone marrow.The MLBM cell population represents 76% of the Lin⁻ population used inprevious examples, whereas only about 37% and 4%, respectively, of Lin⁺and CD31⁻/CD34⁻/CD11b⁻ cell populations from bone marrow expressed CD44(FIG. 26). Accordingly, there is an excellent correlation between CD44expression and the vasculotrophic and neurotrophic activities observedin these three populations, i.e. Lin⁻ cells were the most effectivewhile CD31⁻/CD34⁻/CD11b⁻ cells were consistently the least effective.Using a panel of lineage-specific antibodies, the majority of CD44^(hi)cells were determined to have strongly myeloid characteristics (FIG.27). Similarly, nearly all of the CD44^(hi) bone marrow cells are alsoCD11b⁺ (FIG. 27).

MLBM positively selected using CD11b antibody in Example 12 (CD44^(hi)CD11b⁺) gave activity results similar to those obtained with MLBMisolated using CD44 antibody selection in the vascular targetingexperiments.

The cell surface antigen characteristics of the MLBM cell population ofExample 12 and of the CD44^(lo) CD11b+ cells isolated in Example 12 areshown in Table 3, below. In Table 3, a greater number of plus signs (+)indicates relatively higher expression of the antigen. A minus sign (−)indicates no expression detected.

TABLE 3 Antigen CD44^(hi)/CD11b+ CD44^(lo)/CD11b− CD11a +++ + CD31 + ++CD34 + − alpha 6 ++ − KDR + − Sca-1 + + c-Kit + − CD115 + − CD45R/B220 +++ TER119 − +++ Ly6G&C (GR-1) +++ − Ly6G +++ −

Example 14 Vasculotrophic and Neurotrophic Effects of The MLBM CellPopulation

The MLBM cell population of Example 11 retained the properties of Lin⁻cells in terms of vascular targeting and vasculo- and neurotrophiceffects, while CD44^(lo)BM cells showed little or no activity. Vasculartargeting activity was demonstrated by injecting cells from a GFP⁺ MLBMcell population intravitreally into postnatal day 7 (P7) mice andanalyzing retinas at P14. After labeling blood vessels with GSisolectin, GFP⁺ cells were observed to target the retinal vasculatureand assume a perivascular localization, without evidence ofincorporation. These events were common when using MLBM, but infrequentor absent in eyes treated with CD44^(lo)BM (FIG. 28).

Vasculo- and neurotrophic activity of the MLBM cell population ofExample 11 was evaluated using a mouse model of retinal degeneration asdescribed above for Lin⁻ HSC. The rd1/rd1 mouse shows characteristicfeatures of retinal degenerative disease including photoreceptor deathand atrophy of the deep retinal vasculature. As described above, Lin⁻HSC bone marrow cells preserved the deep retinal vasculature andpartially rescued photoreceptors. The MLBM cell population of thepresent invention performs the same function (FIG. 29).

The oxygen-induced retinopathy model shares features with retinopathy ofprematurity. The pathology associated with this model is significantlyreduced when eyes are treated with cells from the MLBM cell population.The effects of cells from the MLBM cell population in this model weresimilar to those observed using Lin⁻ HSCs described above. Eyes treatedwith cells from the MLBM cell population showed significant reduction inthe two parameters used to quantify the degree of pathology in thismodel: vascular obliteration area and neovascular tuft area. Incontrast, eyes treated with CD44^(lo)BM cells showed no improvement overeyes treated with vehicle controls (FIG. 30).

In addition to targeting retinal vasculature, cells from the MLBM cellpopulation differentiate into macrophage-like (F4/80⁺) cells, penetratethe retina, and take a position closely opposed to the retinal pigmentepithelium (RPE). This localization facilitates the observed vascularand photoreceptor rescue effects of the cells from the MLBM cellpopulation. Furthermore, once in place near the RPE, the cells from theMLBM cell population produce vascular endothelial growth factor (VEGF),as demonstrated by injection of cells from a MLBM cell populationderived from a VEGF-GFP mouse, in which green fluorescent protein (GFP)is expressed upon VEGF gene activation (FIG. 31). Thus, the cells fromthe MLBM cell population appear to be in a VEGF “activated” state. Theintroduced cells from the MLBM cell population appear to recruitendogenous cells of the same type, since both GFP (introduced) and GFP⁻(endogenous) cells were observed in the RPE region. This localizationhas been observed in wild type mice during normal retinal vasculardevelopment, in rescued retinas in the rd1/rd1 mouse and in theoxygen-induced retinopathy model.

Similar vascular targeting results were found for the MLBM cellpopulation of Example 12. FIG. 32 shows that by P20, CD44^(hi) CD11b⁺cells of Example 12 (green) specifically targeted the vasculature (red)when injected at P2, in a manner similar to the CD44-high population ofExample 11. FIG. 33 shows that the CD44^(lo) CD11b⁻ of Example 12 didnot specifically target the vasculature.

The MLBM cell population of the present invention provide an effectiveand versatile treatment for ocular diseases. The cells are readilyisolated from autologous bone marrow, thus minimizing potentialimmunogenicity often observed in cell-based therapies. In addition, theMLBM cell population of the invention can be transfected with usefulgenes for delivering functional genes to the retina.

Example 15 Further Characterization of Bone Marrow Cell Subpopulations

As described in the previous examples, all experiments were performed inaccordance with the NIH Guide for the Care and Use of LaboratoryAnimals, and all experimental procedures were approved by the TSRIAnimal Care and Use Committee. OIR was induced in C57B16 mice accordingto the protocol described above. Post-natal day 7 pups and their motherswere transferred from room air to an environment of 75% oxygen for 5days, and afterwards returned to room air. Oxygen levels were monitoredusing an FDA-approved oxygen analyzer (AX-300, Teledyne AnalyticalInstruments, CA, USA). Under these conditions, large hypovascular areasare formed in the central retina during hyperoxia and abnormalpre-retinal neovascularization occurs after return to normoxia, peakingat around P17 and ultimately resolving (FIG. 37, Panels g-i; FIG. 2,Panels a,c).

Cell Preparation: Mouse bone marrow cell extraction was performedsubstantially as follows: Bone marrow cells were harvested from femursand tibia of actGFP mice and were processed using two different methods.In the first method, mononuclear cells were separated by densitygradient using FICO/LITE LM®(Atlanta Biologicals, Norcross, Ga.) andlabeled with biotin-conjugated lineage antibodies (CD45R/B220, CD3e,Ly-6G/C, CD11b, TER119, Pharmingen, San Diego, Calif.). This wasfollowed by incubation with strepavidin or anti-biotin magnetic beadsand sorting using the MACS cell sorting system (Miltenyi Biotech,Auburn, Calif.) to obtain Lin⁻ HSC populations. In the second method,whole bone marrow was incubated with an antibody directed against CD44,which was conjugated to a fluorescent label. Fluorescence activated cellsorting (FACS) was then used to isolate CD44^(HI) cells (i.e., an MLBMcell population cell population in which as majority of the cellsexpress CD44) and CD44^(HI) cells (i.e., a cell population in which asminority of the cells express CD44).

Bone Marrow Cell Characterization: Further analysis of the cellsubpopulations obtained by the above methods was performed using twoprocedures: (1) two-color flow cytometry in combination with antibodiesagainst various lineage and progenitor cell surface markers, includingCD11a, CD11b, Ly6G/C, CD43, F4/80, CD14, cKit, CD34, α6 integrin, andCD115 (all from Pharmingen, San Diego, Calif.); and (2) gene expressionanalysis using AFFYMETRIX® Mu430 Chips (Affymetrix, Santa Clara, Calif.)using standard methods known in the art. Gene expression was analyzedusing GENESPRING® software (Agilent Technologies, Palo Alto, Calif.).

Intravitreal injection: An eyelid fissure was created by gentledissection to expose the globe in P2-P7 (pre-hyperoxia) mice. In one eyeof each animal, about 150,000 to 250,000 bone marrow-derived cells in0.5 μl vehicle (PBS containing 0.5% BSA and 2 mM EDTA) were injectedinto the vitreous using a Hamilton syringe and a 33 gauge needle(Hamilton, Reno, Nev.). In the contra lateral control eye, anapproximately equal number of control cells or vehicle alone wasinjected, and in some cases no injection was performed at all to observethe natural course of disease. In subsets of experiments, celltransplantation was performed at later ages, between P9 and P12.

Staining of retinal vasculature: Retinas were harvested at P17 forimaging of the vasculature and to localize and characterize the injectedcells. In some cases, animals were anesthetized and intra-cardiacfluorescein-labeled high molecular weight dextran (FITC Dextran, Sigma)was injected prior to dissection of the retinas to visualize patentvessels. In other cases, immunohistological techniques to stain bloodvessels and GFP-expressing cells were used. The retinas were fixed in 4%perfluoroacetic acid (PFA) and methanol, followed by blocking in 20%FBS/20% NGS for one hour at room temperature. This was followed byovernight incubation with isolectin GS-IB4 conjugated to ALEXA® 594 toidentify vessels (Molecular Probes, Eugene, Oreg.). Retinas were laidflat with radial relaxing incisions to obtain whole-mount preparations,or embedded in OCT and cryo-sectioned to obtain cross sections of theretina which are counter-stained with DAPI prior to mounting.

In order to characterize the transplanted cells, immunohistologicaltechniques were used to identify the following cellular markers insubsets of eyes: F4/80 (Caltag, Burlingame, Calif.), CD44, CD31(Pharmingen, San Diego, Calif.), and NG2 (Chemicon, Temecula, Calif.).All retinas were triple stained with lectin, anti-GFP and one of theabove described markers.

Imaging and Image Analysis: Images of the retinal vasculature wereobtained using a RADIANCE® 2100 MP laser scanning confocal microscope(Biorad, Hercules, Calif.). Quantification of vaso-obliteration andneovascularization was carried out as follows: The area of vascularobliteration was measured by carefully outlining the avascular zones inthe central retina of GS lectin-stained retinas and calculating thetotal area using PHOTOSHOP® (Adobe) or VOLOCITY® software (Improvision,Lexington Mass.). Similarly, the area of pre-retinal neovascularization(“tufts”) was calculated by using confocal images focused at thepre-retinal plane and selecting tufts based on pixel intensities (tuftslabel more brightly that normal vasculature). Selected regions were thensummed to generate total area of neovascularization. A T-test was usedto statistically compare the different experimental groups.

Three dimensional images of retinal vasculature and perivascular bonemarrow cells were generated by collecting a z-series of confocal imagesand rendering them into volumes using VOLOCITY® software. It was thenpossible to view retinal vessels in cross section and determine theposition of transplanted bone marrow cells relative to the vascularlumen

Retinal vascular development and the mouse model of oxygen-inducedretinopathy. Normal retinal vascular development in post-natal micegrown under normoxic conditions is shown in FIG. 37, Panels a-f. At postnatal day 2 (P2) only budding superficial vessels are observed occupyinga single plane around the optic disc (FIG. 37, Panels a,b). Over thecourse of the next week, the primary superficial network extends towardsthe periphery, reaching the far periphery at approximately P12 (FIG. 37,Panel c). Between P7-P12, the secondary (deep) plexus develops (FIG. 37,Panel d). By the end of the first month, remodeling occurs in the fullyvascularized retina (FIG. 37, Panel e) along with development of thetertiary (intermediate) layer of vessels, and the adult structure isreached (FIG. 37, Panel f).

In contrast, in the OIR model, exposure to 75% oxygen from P7-P12severely disrupts the normal sequence of events: marked regression ofthe superficial network of vessels that have already formed in thecentral retina occurs, especially along the arteries (FIG. 37, Panel g(P10) and Panels h,i (P17)), and development of the deep plexus isseverely delayed (FIG. 37, Panels k,m, retinal cross sections at P17).Vascular growth, in an abnormal fashion, commences again only afterreturning to normoxic conditions at P12. In essence, these are nowrelatively hypoxic conditions for the severely hypovascular retina. AtP17, some deep vessels can be identified in the periphery, but abnormalpre-retinal neovascular tufts, associated with leak of intravasculardye, can be seen in the mid periphery, at the border between thehypovascular central retina and the more vascularized periphery (FIG.37, Panel h). Over the ensuing days, the superficial and deep vesselsslowly develop in the avascular areas, but neovascular tufts protrudingabove the inner limiting membrane (ILM) of the retina into the vitreousoften persist until P21 or even later. By P25-P30, the retinalvasculature has remodeled and resembles the normal vasculature at thistime.

Injection of hematopoietic progenitor cells prior to hyperoxia promotesvascular repair in the retina following oxygen-inducedvaso-obliteration. Injection of Lin⁻ HSCs of the invention at P2-P7dramatically changed the ability of the retinal vasculature to recoverfollowing hyperoxic exposure (FIG. 37, Panels j,l,n,o, and FIG. 38,Panels b,d,e,f,g). Injection of vehicle alone did not induce suchchanges. In over 50% of cases, fully developed superficial and deepretinal vasculature was seen in Lin⁻ HSC-injected eyes at P17 whilecontra lateral vehicle-injected eyes show large avascular areas andpractically no deep vessels (FIG. 37, Panels 1,n, compared to Panelsh,i,k,m, and to Panel o). In some cases, especially when the injury inthe contra lateral control eye was very severe, recovery was notcomplete by P17 in the Lin⁻ cell-injected eye, but was significantlybetter in the large majority of cases. This comparison between felloweyes in the same animal provides further support for the efficacy of theLin⁻ HSCs, effectively equalizing most other genetic and environmentalfactors.

Vascular obliteration has been an underappreciated feature in thismodel, since most studies have only analyzed pre-retinal neovasculartuft formation in serial retinal sections. Vascular obliteration andtuft formation can be evaluated in the same retina using confocalmicroscopy and digital image analysis (see e.g., FIG. 38, Panels a-d).P17 was selected as the main time point for analysis, because tuftformation is often maximal at this time, while significant vascularobliteration is still present in control eyes. Using the novel method ofcombined analysis, substantial differences between treated and controleyes were identified. Vascular obliteration measured at P17 wassignificantly reduced (over 75% reduction in obliterated area) in Lin⁻treated retinas compared to eyes receiving vehicle alone, or noinjection (FIG. 38, Panel e). No significant difference was observedbetween vehicle injection and no injection in this regard. Similarly,eyes treated with Lin⁻ cells had an approximately 70% reduction inneovascular tuft area compared to vehicle-injected eyes and greater than80% reduction versus non-injected controls (FIG. 38, Panel f). Thus,treatment of eyes with Lin⁻ HSCs had a dramatic effect on the two majorvascular injury and repair parameters of the mouse OIR model, i.e.,simultaneously reducing formation of neovascular tufts whileaccelerating “physiologic” inner-retina revascularization.

Accelerated repair was also observed when treatment was performed duringhyperoxia and upon return to normoxia, but the effect was reduced. Theexperiments described thus far involved injections performed on daysP2-P7, prior to exposure to hyperoxia. To determine whether Lin⁻ cellscould also affect vascular repair if injected later, during thehyperoxia phase of the cycle and upon return to normoxia, injectionswere performed at P9, P11 or P12, and retinas were evaluated at variouslater time points. The results are shown in FIG. 38, Panel g anddemonstrate that injection of Lin⁻ HSCs was effective at acceleratingvascular repair and reducing the area of obliteration even whenadministered during hyperoxia and at P12. The effect, however, appearedto be somewhat attenuated, indicating that maximal efficacy is achievedwhen treatment is performed prior to high oxygen exposure.

Following treatment with Lin⁻ hematopoietic progenitor cells, long termretinal structure and function were well preserved. The long-termeffects and possible side effects of treatment with Lin⁻ HSCs were alsostudied. To this end 12 retinas were taken from mice at 3-6 months ofage that had undergone Lin⁻ cell injection and exposure to hyperoxiaaccording to the established model (FIG. 39). No tumors were observedand the neural retina appeared to be preserved histologically in allcases. The only notable abnormality was an occasional “rosette”formation within the retina, a finding also present in control eyes(FIG. 39, Panels g,h). The retinal vasculature from Lin⁻ HSC-injectedeyes had a normal appearance, and no obvious differences fromnon-injected control retinas were found (FIG. 39, Panels a-f).

Long term persistence of the transplanted cells was also studied. GFP⁺cells were observed in only a small percentage of eyes (10%) indicatingthat the majority of injected cells did not survive beyond severalmonths. When present, surviving cells were often located in closeproximity to the retinal vasculature. Retinal function, as measured byelectroretinographic recordings performed at 17 days to 6 monthspost-transplantation, showed no difference between Lin⁻ HSC-transplantedeyes and normal, non-OIR age-matched controls. To examine thepossibility that transplanted cells may exit the eye and disseminatesystemically, spleens and/or livers from 15 mice were analyzed for thepresence of GFP⁺ cells about 7 to 10 days after injection. Noextra-ocular cells were observed.

Verifying the active cell type: The Lin⁻ population is enriched forCD44^(HI) cells. In an effort to better understand the mechanisms thatmay be active during these processes and to simplify the cell selectionprocedure, an attempt was made to identify a single marker that could beused to isolate active HSCs from the bone marrow. Based oncharacteristics such as involvement in cell migration anddifferentiation, a large panel of candidate bone marrow progenitormarkers was assembled. Using flow cytometry, these markers werescreened, comparing their expression in the active Lin⁻ cells versusthat in control BM cells that were previously shown to be inactive in anumber of experimental systems. CD44 proved to be differentiallyexpressed in these two populations: CD44^(HI) cells were present in asignificantly higher proportion of the Lin⁻ cells (76%) than in thecontrol BM cell population (4%) (FIG. 40, Panel a). As noted above, CD44is a cell surface receptor for hyaluronic acid, and has been shown toparticipate in the regulation of several cellular functions that arebelieved to be important in mediating the rescue effect includingsurvival, migration and differentiation. The distribution of CD44^(HI)cells, being highly prevalent in the active cell population and quiterare in the control cells with reduced activity, indicated that CD44 is,indeed, an effective indicator of activity.

For example, CD44^(HI) cells promote vascular repair in the OIR model,while CD44^(HI) cells do not. The efficacy of CD44^(HI) cells wasverified in the OIR model for their ability to facilitate vascularrepair. Using the same experimental design as that described for Lin⁻cell injections, CD44^(HI) cells were demonstrated to promote retinalvascular repair in this model with efficacy similar to that observedwith Lin⁻ cells (FIG. 40, Panels b,c). In contrast, CD4410 cells had nopositive effect on repair. It is of value to point out that often few orno injected cells were observed within the retinas of CD44^(LO)-treatedanimals, suggesting that these cells have reduced ability to survive inthe vitreous and/or migrate into the retina. It is not known whether theCD44^(HI) cells are the only active bone marrow sub-population or one ofothers that have this activity.

CD44^(HI) cells express genes and markers suggestive of myeloid origin.Further characterization of the CD44^(HI) population was performed bylarge-scale expression analysis and by antibody labeling of Lin⁻ andprogenitor-specific markers followed by flow cytometry (FIG. 41, andFIG. 44). Both methods revealed that CD44^(HI) cells have an expressionprofile suggestive of myeloid origin. Strong expression of CD11a, CD11b,and Ly6G/C was observed on these cells at the protein level, while lessintense positive labeling was detected for F4/80, CD14, cKit and CD115by flow cytometry. Several myeloid-specific genes including CD204,CD114, CD33 and CD115 were highly expressed on expression analysis ascompared with CD4410 cells (FIG. 44). In contrast, at the protein level,the CD44^(LO) population had significant expression of Ter119 and CD45RB220, which are markers of erythroblasts/erythrocytes and B cells,respectively. On the expression array, a number of genes associated withlymphocytes were highly expressed in CD44^(LO) as compared withCD44^(HI) cells including CD19, CD79a and CD22 (FIG. 44). Thus, analysisat the transcriptional and protein levels identifies the activeCD44^(HI) population as primarily myeloid in origin while the inactiveCD44^(LO) cells are largely lymphoid.

Analysis of transplanted cells in situ—evidence for differentiation:Having more clearly defined the population of active cells from the bonemarrow, the fate of these cells after introduction into the eye wasinvestigated. To this end, CD44^(HI)-injected retinas from the OIR modelwere analyzed by immunohistochemistry with various markers. The vastmajority of introduced cells selectively targeted the retinalvasculature and assumed a perivascular localization, often formingelongated structures tightly associated with host vessels (FIG. 42,Panel a). Using antibodies against CD31 and NG2, these markers were notdetected on the GFP-expressing perivascular bone marrow cells,suggesting that these cells are not differentiating into endothelialcells or pericytes, respectively. In addition, the transplanted cellsdid not appear to form any portion of the vessel lumen (FIG. 42, Panelb), thus demonstrating that these cells are unlikely to bedifferentiating into endothelial cells. In contrast, themacrophage/microglia marker F4/80 labeled many, but not all,perivascular GFP⁺ cells in CD44^(HI)-treated eyes (FIG. 43, Panels d-i).These introduced F4/80⁺ cells had an appearance very similar toendogenous perivascular cells which also labeled with F4/80 (FIG. 43,Panels a-c), suggesting that the transplanted cells were assuming anidentity similar to native cells in the OIR model.

One of the possible advantages of cell therapy, particularly incomparison to conventional pharmaceutical treatment, is the potential ofthe cells to respond to local cues and undergo modification in changingenvironments. Transplanted cells at P17 (10 days after injection) thathad targeted the retinal vasculature and assumed a perivascular locationwere observed to have down-regulated CD44 to undetectable levels (FIG.43, Panels j-o). Cells that were not associated with the vasculatureretained expression of CD44, however. Thus, sub-populations of implantedcells that were originally selected by FACS on the basis of high CD44expression down-regulated this receptor in-vivo, which correlated withthe location of the cells in the retina. This suggests that theintroduced cells do, indeed, undergo selective changes (differentiation)within the environment of the eye.

The results detailed above indicate that cell-based therapy can be usedto treat ROP, and other ischemic retinopathies. The results observed inthe mouse model indicate that this approach is efficacious in reducingthe vascular pathology associated with high oxygen exposure and showslittle or no toxicity. The advantage of using cell therapy, as opposedto single factor therapy, may lie in the ability of the cell to adaptand respond to a changing environment. The evolution from single factortherapeutics, to combinations of drugs and interventions, to theselection and delivery of sophisticated, adaptable cells that canorchestrate and conduct a complicated sequence of responses whileinteracting with the host tissue is an exciting new concept. In thisrespect, the present invention provides a “paradigm shift” in theapproach to ischemic retinopathies/vasculopathies, i.e., emphasizinghealing and stabilization instead of inhibition and obliteration.

The isolated MLBM cell populations of the invention target the retinalvasculature, can be used to deliver angiostatic agents, and havevasculo- and neurotrophic effects in models of retinal degeneration. Inthe present study, specific subpopulations of Lin⁻ isolated MLBM cellpopulations are highly effective in accelerating the repair of OIR.Interestingly, the active cells express markers that suggest that theyare of myeloid origin, and perhaps undergo differentiation andmodification following transplantation.

The use of cell therapy to promote vascularization has been spearheadedby the field of cardiology with the goal of collateralizing infarctedarteries. A substantial amount of evidence indicates that certain bonemarrow cells are effective at improving perfusion and cardiac function.It is not yet clear, however, which cell type(s) are responsible for theobserved effects. Numerous studies investigating the potential role ofbone marrow-derived endothelial progenitor cells (EPCs) have concludedthat these cells are present in new or collateral vessels, but the smallnumber of incorporated cells reported in some of these studies raisesquestions regarding their importance. Additionally, heterogeneous bonemarrow populations, such as mononuclear cells or unfractionated cells,which contain very small numbers of stem cells and/or EPCs, can alsosignificantly enhance collateral development, suggesting othermechanisms beyond direct incorporation into vessels are at work. Whilenot intending to be bound by theory, it is possible that these cellsplay a supportive, paracrine role, by which factors secreted from themact to optimize the conditions for the host vasculature. Many bonemarrow subpopulations have been shown to be a source of angiogenicfactors, and monocytic cells are known to secrete a variety of suchfactors. Thus, the potential exists for bone marrow cells to serve in aparacrine fashion, complementing the role of EPCs in collateral vesselformation and interacting with the host immune system.

Although the precise mechanisms at work in this system are not yetclear, significant progress has been made in terms of understanding thenature of the functional bone marrow cells. With the identification ofan active myeloid population within bone marrow, as provided by thecells of the present invention, some suggestions regarding mechanism canbe made. Myeloid cells, notably monocytes and macrophages, haveestablished abilities to influence blood vessel growth through secretionof angiogenic growth factors. In addition, macrophages have been shownto be more tolerant of hypoxia than other cells types and respond to lowoxygen conditions by secretion of angiogenic factors. Thus, introducingmyeloid progenitors into ischemic retinas could provide a cell that canwithstand hypoxic conditions and can promote vascular repair in aparacrine manner. The presence of host-derived F4/80⁺ perivascular cellsin the OIR retina suggests that this type of cell has a role in theprocess, and perhaps the delivery of a large pool of similar cells (ortheir progenitors) by direct transplantation into the eye augments thiseffect. This scenario highlights the paradoxical observation that, asobserved in the present studies, injection of cell populations of thepresent invention promotes revascularization of the retina whilesuppressing pre-retinal neovascularization. Although the basis for thisis not yet fully known, it is possible that accelerated “physiologic”revascularization may reduce the hypoxia experienced by the retina suchthat ischemia-stimulated neovascular tufts do not form to the samedegree.

The idea of myeloid-like cell support of vessel growth may haverelevance to some earlier work relating to the rd1 and rd10 mouse modelsof retinal degeneration. Injected myeloid progenitors could act tomaintain the deep retinal vasculature through secreted factors andprevent the vessel degeneration that is observed in these models. Somemacrophage-secreted angiogenic factors, such as bFGF, have demonstratedneurotrophic activity as well. Thus, the observed reduced photoreceptordeath upon injection of the cell populations of the present invention inrd mice could be mediated though a paracrine mechanism, in whichneurotrophic factors are produced by the transplanted bonemarrow-derived myeloid cells. In support of this mechanism, the presentstudies indicate that THE isolated MLBM cell populations of theinvention are capable of vascular and neuronal rescue in the rd modelwith efficacy similar to that observed upon injection of isolated MLBMcells.

In a clinical treatment for ROP, fetal cord blood cells are harvestedduring the birth of a high risk premature infant, the cells are thensorted to enrich for the specific subpopulation which mediates therescue effect, and these autologous progenitor cells can then beinjected into the eye of the infant.

One of the main current limitations for the use of cell therapy is thefact that in many cases the exact molecular mechanisms of action are notyet clear, and in fact these mechanisms may differ between models.However, this may actually be the greatest advantage of cell-basedtherapies, i.e., the ability to respond in a different way and with awide repertoire to changing conditions and cues. This is true not onlybetween different experimental systems and challenges, but alsotemporally within one system. In other words, such cells may besecreting certain factors at one time point and different factors atanother and ultimately, if the need for them subsides, may cease actingaltogether. This is something that current chemical-based drug therapiescannot do, and is based on the fact that cells fundamentally use andrespond to feedback. The modification of cellular markers in thetransplanted cells in vivo observed in the present study supports thisconcept.

Example 16 MLBM Cells Differentiate Into Cells With MicroglialCharacteristics

Analysis of retinas following injection of CD44^(HI) cells indicatesthat the CD44^(HI) population of bone marrow cells are differentiatinginto microglia after injection into the eye. Microglia are the residentmyeloid population in the retina and express characteristic markersincluding CD11b and F4/80. These cells are also distinguished by theirramified (branched) morphology and assume perivascular localization. Thelocalization, morphology and surface marker expression of CD44hi cellsat various points after injection into eyes has been analyzed. It isobserved that injected CD44^(HI) GFP⁺ cells display all of the describedcharacteristics of endogenous retinal microglia (FIG. 45). Panels A andB in FIG. 45 show that injected CD44^(HI) cells express CD11b and F4/80and have morphology and perivascular localization similar to endogenousmicroglia. Panel C provides 3D imaging analysis that demonstrates thatinjected CD44^(HI) cells localize in the perivascular region. Panel Dshows a high magnification view of the morphology of injected CD44^(HI)cells.

Example 17 Isolation of MLBM Cells By Negative Selection

It is desirable for the purposes of experimentation and clinicalapplications to inject cells that are free of surface-bound selectionagents, such as antibodies and/or magnetic beads. One way of achievingthis goal is to utilize a negative selection strategy to isolateCD44^(HI) cells. Through characterization of the surface markerexpression profiles of the CD44^(HI) and CD44^(LO) cell populationsdescribed herein, it has been discovered that CD44^(LO) cells displayedhigh expression of Ter119 and CD45RB220, markers of erythroid cells andB cells, respectively. Antibodies against these markers, with theaddition of the T cell marker CD3e, efficiently labeled the CD44^(LO)population and allowed for their removal via magnetic or FACSseparation, leaving “untouched” CD44^(HI) cells as the product. Cellsseparated by FACS using this strategy show the typical functionalcharacteristics of the MLBM cell populations of the present invention(FIG. 46).

FIG. 46, Panel A shows that depletion of mouse bone marrow by MACS usingantibodies selective for CD45R/B220, TER119, and CD3e yields apopulation of cells that are greater than 90 percent CD44^(HI) cells.Panel B shows the negative fraction (CD44^(HI) population) isessentially free from CD45R/B220, TER119, and CD3e cells. Panel C showsnegatively selected CD44^(HI) cells retain retinal targeting anddifferentiation capabilities.

Numerous variations and modifications of the embodiments described abovemay be effected without departing from the spirit and scope of the novelfeatures of the invention. No limitations with respect to the specificembodiments illustrated herein are intended or should be inferred.

1. A method for isolating a myeloid-like bone marrow cell populationfrom bone marrow by negative cell marker selection, the methodcomprising contacting a plurality of bone marrow cells with antibodiesspecific for Ter119, CD45RB220, and CD3e; removing cells from theplurality of bone marrow cells that immunoreact with Ter119, CD45RB220,and CD3e antibodies from the plurality of bone marrow cells; andrecovering myeloid-like bone marrow cells that are deleted in Ter119,CD45RB220, and CD3e-expressing cells; wherein a majority of therecovered cells express CD44.
 2. A myeloid-like bone marrow cellpopulation prepared by the method of claim
 1. 3. The myeloid-like bonemarrow cell population of claim 2 wherein greater than 90 percent of thecells in the population express CD44.